Limited efficacy of a commercial microbial inoculant for improving growth and physiological performance of native plant species.

Soil microbial inoculants are increasingly being explored as means to improve soil conditions to facilitate ecological restoration. In southwestern Western Australia, highly biodiverse Banksia woodland plant communities are increasingly threatened by various factors including climate change, land development and mining. Banksia woodland restoration is necessary to conserve this plant community. The use of microbial inoculation in Banksia woodland restoration has not yet been investigated. Here, we evaluated the efficacy of a commercial microbial inoculant (GOGO Juice, Neutrog Australia Pty Ltd) for improving the performance of 10 ecologically diverse Banksia woodland plant species in a pot experiment. Plants were subjected to one of two watering regimes (well-watered and drought) in combination with microbial inoculation treatments (non-inoculated and inoculated). Plants were maintained under these two watering treatments for 10 weeks, at which point plants in all treatments were subjected to a final drought period lasting 8 weeks. Plant performance was evaluated by plant biomass and allocation, gas exchange parameters, foliar carbon and nitrogen and stable isotope (δ15N and δ13C) compositions. Plant xylem sap phytohormones were analysed to investigate the effect of microbial inoculation on plant phytohormone profiles and potential relationships with other observed physiological parameters. Across all investigated plant species, inoculation treatments had small effects on plant growth. Further analysis within each species revealed that inoculation treatments did not result in significant biomass gain under well-watered or drought-stressed conditions, and effects on nitrogen nutrition and photosynthesis were variable and minimal. This suggests that the selected commercial microbial inoculant had limited benefits for the tested plant species. Further investigations on the compatibility between the microorganisms (present in the inoculant) and plants, timing of inoculation, viability of the microorganisms and concentration(s) required to achieve effectiveness, under controlled conditions, and field trials are required to test the feasibility and efficacy in actual restoration environments.

Cobalt and Titanium Alleviate the Methylglyoxal-Induced Oxidative Stress in Pennisetum divisum Seedlings under Saline Conditions.

Salinity is considered to be a global problem and a severe danger to modern agriculture since it negatively impacts plants' growth and development at both cellular- and whole-plant level. However, cobalt (Co) and titanium (Ti), multifunctional non-essential micro-elements, play a crucial role in improving plant growth and development under salinity stress. In the current study, Co and Ti impact on the morphological, biochemical, nutritional, and metabolic profile of Pennisetum divisum plants under three salinity levels which were assessed. Two concentrations of Co (Co-1; 15.0 mg/L and Co-2; 25.0 mg/L), and two concentrations of Ti (Ti-1; 50.0 mg/L and Ti-2; 100.0 mg/L) were applied as foliar application to the P. divisum plants under salinity (S1; 200 mM, S2; 500 mM, and S3; 1000 mM) stress. The results revealed that various morphological, biochemical, and metabolic processes were drastically impacted by the salinity-induced methylglyoxal (MG) stress. The excessive accumulation of salt ions, including Na+ (1.24- and 1.21-fold), and Cl- (1.53- and 1.15-fold) in leaves and roots of P. divisum, resulted in the higher production of MG (2.77- and 2.95-fold) in leaves and roots under severe (1000 mM) salinity stress, respectively. However, Ti-treated leaves showed a significant reduction in ionic imbalance and MG concentrations, whereas considerable improvement was shown in K+ and Ca2+ under salinity stress, and Co treatment showed downregulation of MG content (26, 16, and 14%) and improved the antioxidant activity, such as a reduction in glutathione (GSH), oxidized glutathione (GSSG), Glutathione reductase (GR), Glyoxalase I (Gly I), and Glyoxalase II (Gly II) by up to 1.13-, 1.35-, 3.75-, 2.08-, and 1.68-fold under severe salinity stress in P. divisum roots. Furthermore, MG-induced stress negatively impacted the metabolic profile and antioxidants activity of P. divisum's root and leaves; however, Co and Ti treatment considerably improved the biochemical processes and metabolic profile in both underground and aerial parts of the studied plants. Collectively, the results depicted that Co treatment showed significant results in roots and Ti treatment presented considerable changes in leaves of P. divism under salinity stress.

Metabolic Processes and Biological Macromolecules Defined the Positive Effects of Protein-Rich Biostimulants on Sugar Beet Plant Development.

Protein-based biostimulants (PBBs) have a positive effect on plant development, although the biological background for this effect is not well understood. Here, hydrolyzed wheat gluten (HWG) and potato protein film (PF) in two levels (1 and 2 g/kg soil) and in two different soils (low and high nutrient; LNC and HNC) were used as PBBs. The effect of these PBBs on agronomic traits, sugars, protein, and peptides, as well as metabolic processes, were evaluated on sugar beet in comparison with no treatment (control) and treatment with nutrient solution (NS). The results showed a significant growth enhancement of the plants using HWG and PF across the two soils. Sucrose and total sugar content in the roots were high in NS-treated plants and correlated to root growth in HNC soil. Traits related to protein composition, including nitrogen, peptide, and RuBisCO contents, were enhanced in PBB-treated plants (mostly for HWG and PF at 2 g/kg soil) by 100% and >250% in HNC and LNC, respectively, compared to control. The transcriptomic analysis revealed that genes associated with ribosomes and photosynthesis were upregulated in the leaf samples of plants treated with either HWG or PP compared to the control. Furthermore, genes associated with the biosynthesis of secondary metabolites were largely down-regulated in root samples of HWG or PF-treated plants. Thus, the PBBs enhanced protein-related traits in the plants through a higher transcription rate of genes related to protein- and photosynthesis, which resulted in increased plant growth, especially when added in certain amounts (2 g/kg soil). However, sucrose accumulation in the roots of sugar beet seemed to be related to the easy availability of nitrogen.

Biochar-Mediated Control of Metabolites and Other Physiological Responses in Water-Stressed Leptocohloa fusca.

We investigated biochar-induced drought tolerance in Leptocohloa fusca (Kallar grass) by exploring the plant defense system at physiological level. L. fusca plants were exposed to drought stress (100%, 70%, and 30% field capacity), and biochar (BC), as an organic soil amendment was applied in two concentrations (15 and 30 mg kg-1 soil) to induce drought tolerance. Our results demonstrated that drought restricted the growth of L. fusca by inhibiting shoot and root (fresh and dry) weight, total chlorophyll content and photosynthetic rate. Under drought stress, the uptake of essential nutrients was also limited due to lower water supply, which ultimately affected metabolites including amino and organic acids, and soluble sugars. In addition, drought stress induced oxidative stress, which is evidenced by the higher production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide ion (O2-), hydroxyl ion (OH-), and malondialdehyde (MDA). The current study revealed that stress-induced oxidative injury is not a linear path, since the excessive production of lipid peroxidation led to the accumulation of methylglyoxal (MG), a member of reactive carbonyl species (RCS), which ultimately caused cell injury. As a consequence of oxidative-stress induction, the ascorbate-glutathione (AsA-GSH) pathway, followed by a series of reactions, was activated by the plants to reduce ROS-induced oxidative damage. Furthermore, biochar considerably improved plant growth and development by mediating metabolites and soil physio-chemical status.

Is the NH4 +-induced growth inhibition caused by the NH4 + form of the nitrogen source or by soil acidification?

Soil acidification often occurs when the concentration of ammonium (NH4 +) in soil rises, such as that observed in farmland. Both soil acidification and excess NH4 + have serious adverse effects on crop growth and food production. However, we still do not know which of these two inhibitors has a greater impact on the growth of crops, and the degree of their inhibitory effect on crop growth have not been accurately evaluated. 31 wheat cultivars originating in various areas of China were planted under 5 mM sole NH4 + (ammonium nitrogen, AN) or nitrate nitrogen in combined with two pH levels resembling acidified conditions (5.0 and 6.5). The results showed that the shoots and roots biomass were severely reduced by AN in both and these reduction effects were strengthened by a low medium pH. The concentration of free NH4 + and amino acids, the glutamine synthetase activity were significantly higher, but the total soluble sugar content was reduced under NH4 + conditions, and the glutamine synthetase activity was reduced by a low medium pH. Cultivar variance was responsible for the largest proportion of the total variance in plant dry weight, leaf area, nodal root number, total root length and root volume; the nitrogen (N) form explains most of the variation in N and C metabolism; the effects of pH were the greatest for plant height and root average diameter. So, soil acidification and excess NH4 + would cause different degrees of inhibition effects on different plant tissues. The findings are expected to be useful for applying effective strategies for reducing NH4 + stress in the field.

Growing together gives more rice and aquatic food.

Allowing aquatic organisms to grow in rice fields - a practice called co-culture - increases rice yields while maintaining soil fertility and reducing weeds.

Harnessing Synergistic Biostimulatory Processes: A Plausible Approach for Enhanced Crop Growth and Resilience in Organic Farming.

Demand for organically grown food crops is rising substantially annually owing to their contributions to human health. However, organic farm production is still generally lower compared to conventional farming. Nutrient availability, content consistency, uptake, assimilation, and crop responses to various stresses were reported as critical yield-limiting factors in many organic farming systems. In recent years, plant biostimulants (BSs) have gained much interest from researchers and growers, and with the objective of integrating these products to enhance nutrient use efficiency (NUE), crop performance, and delivering better stress resilience in organic-related farming. This review gave an overview of direct and indirect mechanisms of microbial and non-microbial BSs in enhancing plant nutrient uptake, physiological status, productivity, resilience to various stressors, and soil-microbe-plant interactions. BSs offer a promising, innovative and sustainable strategy to supplement and replace agrochemicals in the near future. With greater mechanistic clarity, designing purposeful combinations of microbial and non-microbial BSs that would interact synergistically and deliver desired outcomes in terms of acceptable yield and high-quality products sustainably will be pivotal. Understanding these mechanisms will improve the next generation of novel and well-characterized BSs, combining microbial and non-microbial BSs strategically with specific desired synergistic bio-stimulatory action, to deliver enhanced plant growth, yield, quality, and resilience consistently in organic-related cultivation.

Higher Atmospheric CO2 Levels Favor C3 Plants Over C4 Plants in Utilizing Ammonium as a Nitrogen Source.

Photosynthesis of wheat and maize declined when grown with NH4 + as a nitrogen (N) source at ambient CO2 concentration compared to those grown with a mixture of NO3 - and NH4 +, or NO3 - as the sole N source. Interestingly, these N nutritional physiological responses changed when the atmospheric CO2 concentration increases. We studied the photosynthetic responses of wheat and maize growing with various N forms at three levels of growth CO2 levels. Hydroponic experiments were carried out using a C3 plant (wheat, Triticum aestivum L. cv. Chuanmai 58) and a C4 plant (maize, Zea mays L. cv. Zhongdan 808) given three types of N nutrition: sole NO3 - (NN), sole NH4 + (AN) and a mixture of both NO3 - and NH4 + (Mix-N). The test plants were grown using custom-built chambers where a continuous and desired atmospheric CO2 (C a ) concentration could be maintained: 280 µmol mol-1 (representing the pre-Industrial Revolution CO2 concentration of the 18th century), 400 µmol mol-1 (present level) and 550 µmol mol-1 (representing the anticipated futuristic concentration in 2050). Under AN, the decrease in net photosynthetic rate (P n ) was attributed to a reduction in the maximum RuBP-regeneration rate, which then caused reductions in the maximum Rubisco-carboxylation rates for both species. Decreases in electron transport rate, reduction of electron flux to the photosynthetic carbon [Je(PCR)] and electron flux for photorespiratory carbon oxidation [Je(PCO)] were also observed under AN for both species. However, the intercellular (C i ) and chloroplast (C c ) CO2 concentration increased with increasing atmospheric CO2 in C3 wheat but not in C4 maize, leading to a higher Je(PCR)/ Je(PCO) ratio. Interestingly, the reduction of P n under AN was relieved in wheat through higher CO2 levels, but that was not the case in maize. In conclusion, elevating atmospheric CO2 concentration increased C i and C c in wheat, but not in maize, with enhanced electron fluxes towards photosynthesis, rather than photorespiration, thereby relieving the inhibition of photosynthesis under AN. Our results contributed to a better understanding of NH4 + involvement in N nutrition of crops growing under different levels of CO2.

Gigantic chloroplasts, including bizonoplasts, are common in shade-adapted species of the ancient vascular plant family Selaginellaceae.

PREMISE: Unique among vascular plants, some species of Selaginella have single giant chloroplasts in their epidermal or upper mesophyll cells (monoplastidy, M), varying in structure between species. Structural variants include several forms of bizonoplast with unique dimorphic ultrastructure. Better understanding of these structural variants, their prevalence, environmental correlates and phylogenetic association, has the potential to shed new light on chloroplast biology unavailable from any other plant group. METHODS: The chloroplast ultrastructure of 76 Selaginella species was studied with various microscopic techniques. Environmental data for selected species and subgeneric relationships were compared against chloroplast traits. RESULTS: We delineated five chloroplast categories: ME (monoplastidy in a dorsal epidermal cell), MM (monoplastidy in a mesophyll cell), OL (oligoplastidy), Mu (multiplastidy, present in the most basal species), and RC (reduced or vestigial chloroplasts). Of 44 ME species, 11 have bizonoplasts, cup-shaped (concave upper zone) or bilobed (basal hinge, a new discovery), with upper zones of parallel thylakoid membranes varying subtly between species. Monoplastidy, found in 49 species, is strongly shade associated. Bizonoplasts are only known in deep-shade species (<2.1% full sunlight) of subgenus Stachygynandrum but in both the Old and New Worlds. CONCLUSIONS: Multiplastidic chloroplasts are most likely basal, implying that monoplastidy and bizonoplasts are derived traits, with monoplastidy evolving at least twice, potentially as an adaptation to low light. Although there is insufficient information to understand the adaptive significance of the numerous structural variants, they are unmatched in the vascular plants, suggesting unusual evolutionary flexibility in this ancient plant genus.

The Bacterial Microbiome Associated With Arid Biocrusts and the Biogeochemical Influence of Biocrusts Upon the Underlying Soil.

Biocrusts are aggregated crusts that exist on the soil surface of arid environments. They are complex microbial communities comprised of cyanobacteria, lichens, mosses, algae and fungi. Recently, biocrusts have gained significant attention due to their ubiquitous distribution and likely important ecological roles, including soil stabilization, soil moisture retention, carbon (C) and nitrogen (N) fixation, as well as microbial engineers for semi-arid ecosystem restoration. Here, we collected three co-occurring types of biocrust (Cyanobacterial crust, Crustose lichen, and Foliose lichen) and their underlying soil from arid zones within Western Australia. Bacterial microbiome composition was determined through 16S rRNA gene amplicon sequencing to assess the extent of microbiome selection within the crusts versus underlying soil and biogeochemical measures performed to determine whether the crusts had significant impact upon the underlying soil for nutrient input. We determined that the bacterial communities of native biocrusts are distinct from those in their underlying soil, where dominant bacterial taxa differed according to crust morphologies. δ15N revealed that N-fixation appeared most evident in Foliose lichen crust (1.73 ± 1.04‰). Consequently, depending upon the crust type, biocrusts contained higher concentrations of organic C (2 to 50 times), total N (4 to 16 times) and available ammonium (2 to 4 times), though this enrichment did not extend to the soils underneath them. These findings demonstrate that biocrust communities are seemingly islands of biological activity in an arid landscape, uniquely different from their surrounding and underlying soil.

Mechanisms underlying enhanced Cd translocation and tolerance in roots of Populus euramericana in response to nitrogen fertilization.

A pot experiment was conducted to evaluate how nitrogen (N) availability influences cadmium (Cd) absorption, translocation and stress tolerance in roots of Populus euramericana. Seedling growth was sensitive to N deficiency, but it was unaltered by Cd exposure. Cadmium absorption by roots was promoted by N deficiency, resulting in a higher root Cd concentration compared to the N-sufficient condition. Fine-root length was tightly correlated (R2 = 0.73) with Cd concentration in roots, indicating that vigorous fine-root proliferation under N deficiency contributed to active absorption and accumulation of Cd in roots. Despite accumulation in roots, Cd translocation from roots to shoots was less active under N deficiency compared to N sufficiency. This was related to elevated glutathione reductase (GR) activity and glutathione (GSH) levels in roots after N application, which may not only promote antioxidant defence, but also facilitate the formation of GSH-Cd complexes that are uploaded into root cylinders. Nitrogen application also promoted antioxidant defense in roots via increased production of phytohormones and the level of enzymatic and non-enzymatic antioxidants. Transcript levels for genes responsible for antioxidant defense, Cd detoxification and Cd uploading were increased in roots by N application. The N-stimulated Cd tolerance, detoxification and uploading in roots are factors likely to promote Cd translocation from roots to shoots, which may enhance the biological capacity of this poplar species for phytoremediation of Cd pollution.

Leaf structure affects a plant's appearance: combined multiple-mechanisms intensify remarkable foliar variegation.

The presence of foliar variegation challenges perceptions of leaf form and functioning. But variegation is often incorrectly identified and misinterpreted. The striking variegation found in juvenile Blastus cochinchinensis (Melastomataceae) provides an instructive case study of mechanisms and their ecophysiological implications. Variegated (white and green areas, vw and vg) and non-variegated leaves (normal green leaves, ng) of seedlings of Blastus were compared structurally with microtechniques, and characterized for chlorophyll content and fluorescence. More limited study of Sonerila heterostemon (Melastomataceae) and Kaempferia pulchra (Zingiberaceae) tested the generality of the findings. Variegation in Blastus combines five mechanisms: epidermal, air space, upper mesophyll, chloroplast and crystal, the latter two being new mechanisms. All mesophyll cells (vw, vg, ng) have functional chloroplasts with dense thylakoids. The vw areas are distinguished by flatter adaxial epidermal cells and central trichomes containing crystals, the presence of air spaces between the adaxial epidermis and a colorless spongy-like upper mesophyll containing smaller and fewer chloroplasts. The vw area is further distinguished by having the largest spongy-tissue chloroplasts and fewer stomata. Both leaf types have similar total chlorophyll content and similar  F v/F m (maximum quantum yield of PSII), but vg has significantly higher F v/F m than ng. Variegation in Sonerila and Kaempferia is also caused by combined mechanisms, including the crystal type in Kaempferia. This finding of combined mechanisms in three different species suggests that combined mechanisms may occur more commonly in nature than current understanding. The combined mechanisms in Blastus variegated leaves represent intricate structural modifications that may compensate for and minimize photosynthetic loss, and reflect changing plant needs.

An invasive plant promotes its arbuscular mycorrhizal symbioses and competitiveness through its secondary metabolites: indirect evidence from activated carbon.

Secondary metabolites released by invasive plants can increase their competitive ability by affecting native plants, herbivores, and pathogens at the invaded land. Whether these secondary metabolites affect the invasive plant itself, directly or indirectly through microorganisms, however, has not been well documented. Here we tested whether activated carbon (AC), a well-known absorbent for secondary metabolites, affect arbuscular mycorrhizal (AM) symbioses and competitive ability in an invasive plant. We conducted three experiments (experiments 1-3) with the invasive forb Solidago canadensis and the native Kummerowia striata. Experiment 1 determined whether AC altered soil properties, levels of the main secondary metabolites in the soil, plant growth, and AMF communities associated with S. canadensis and K. striata. Experiment 2 determined whether AC affected colonization of S. canadensis by five AMF, which were added to sterilized soil. Experiment 3 determined the competitive ability of S. canadensis in the presence and absence of AMF and AC. In experiment 1, AC greatly decreased the concentrations of the main secondary metabolites in soil, and the changes in concentrations were closely related with the changes of AMF in S. canadensis roots. In experiment 2, AC inhibited the AMF Glomus versiforme and G. geosporum but promoted G. mosseae and G. diaphanum in the soil and also in S. canadensis roots. In experiment 3, AC reduced S. canadensis competitive ability in the presence but not in the absence of AMF. Our results provided indirect evidence that the secondary metabolites (which can be absorbed by AC) of the invasive plant S. canadensis may promote S. canadensis competitiveness by enhancing its own AMF symbionts.

Rhizobium-induced elevation in xylem cytokinin delivery in pigeonpea induces changes in shoot development and leaf physiology.

Inoculation with Rhizobium strain IC3342 induces in pigeonpea (Cajanus cajan (L) Millsp.) a leaf curl syndrome and elevated cytokinin levels in the xylem sap. High nitrogen (N) nutrition was found to inhibit onset of the syndrome which could then be induced by N-free nutrient after development of seven trifoliate leaves. This provided a new system to study the role of xylem cytokinin in shoot development and yielded plants suitable for determining the rate of delivery of xylem cytokinin to the shoot which for IC3342-inoculated plants was found to be three times that of control plants. Relative to leaves of control plants, the non-curled leaves of these IC3342 plants exhibited higher nitrogen and chlorophyll content and greater photosynthetic rate and stomatal conductance. Induction of the syndrome increased leaf thickness in developing leaves but not in expanded leaves already formed. Diameter of stems and number of laterals were also increased markedly by IC3342 inoculation which in addition induced leaf hyponasty. Exogenous cytokinins when applied directly to control leaves induced leaf curl and increased leaf thickness. The present studies are discussed in relation to the role of xylem cytokinins in plant development and especially the release of lateral buds from apical dominance.

Legumes can increase cadmium contamination in neighboring crops.

Legumes are widely used in many cropping systems because they share their nitrogen fixation products and phosphorus mobilization activities with their neighbors. In the current study, however, we showed that co-cultivation with legumes increased cadmium (Cd) contamination in the adjacent crops. Both field and mesocosm experiments indicated that legumes increased Cd levels in edible parts and shoots of four neighboring crops and five maize varieties tested, regardless of the Cd levels in the soil. This enhanced Cd accumulation in crops was attributed to root interactions that alter the rhizosphere environment. Co-cultivation with legumes reduced soil pH, which somewhat increased the exchangeable forms of Cd. Our results have demonstrated the inevitable increases in Cd levels of crops as a direct result of co-cultivation with legumes even under situations when these levels are below the permissible threshold. With this new revelation, we need to consider carefully the current cropping systems involving legumes and perhaps to re-design the current and future cropping systems in view of avoiding food contamination by Cd.

Surfactant-assisted pressurized liquid extraction for determination of flavonoids from Costus speciosus by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC), a mode of capillary electrophoresis (CE), is considered an efficient analytical technique allowing for the reduction of organic solvent consumption during the experimental procedure. However, during sample preparation of natural products, the usage of large amount of organic solvent is generally unavoidable. In this article, therefore, a fast, simple, efficient, highly automatic and organic solvent-free sample preparation method, namely surfactant-assisted pressurized liquid extraction (PLE), was developed for the extraction of flavonoids in Costus speciosus flowers before MEKC analysis. The various experimental parameters such as the type and concentration of surfactant, and extraction time were evaluated systematically. Under the optimized conditions, the extraction efficiencies of surfactant-assisted PLE methods were comparable with Soxhlet extraction using organic solvent. The combination of surfactant-assisted PLE and MEKC was shown to be a green, rapid and effective approach for extraction and analysis of flavonoids in C. speciosus flowers.

A novel method of protein extraction from yeast using ionic liquid solution.

As the first attempt, ionic liquid solutions have been employed for direct extraction of proteins from yeast cells. Compared with effects of 21 different ionic liquid solutions on the extraction efficiency, 3-(dimethylamino)-1-propylaminium formate ([DMAPA]FA) was selected as the suitable ionic liquid solution. As this ionic liquid can be easily removed under vacuum, contamination by the chemical noise can be effectively reduced. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (2-DE) were employed to separate numerous proteins, and the results indicated that the chemical properties of target proteins remained unchanged during the extraction process. Furthermore, extracted proteins were applicable to the standard method for Western blotting which showed proteins maintain immunoreactivity and biological functions. These investigations indicated that the ionic liquid [DMAPA]FA is a promising reagent for protein extraction in yeast cells.

The chemical composition and biological properties of coconut (Cocos nucifera L.) water.

Coconut water (coconut liquid endosperm), with its many applications, is one of the world's most versatile natural product. This refreshing beverage is consumed worldwide as it is nutritious and beneficial for health. There is increasing scientific evidence that supports the role of coconut water in health and medicinal applications. Coconut water is traditionally used as a growth supplement in plant tissue culture/micropropagation. The wide applications of coconut water can be justified by its unique chemical composition of sugars, vitamins, minerals, amino acids and phytohormones. This review attempts to summarise and evaluate the chemical composition and biological properties of coconut water.

Separation of cytokinin isomers with a partial filling-micellar electrokinetic chromatography-mass spectrometry approach.

A new method based on partial filling-MEKC (PF-MEKC) directly coupled to ESI-MS was developed for the simultaneous separation and determination of 13 structurally similar cytokinins, including various geometric and positional isomers of cytokinins. On the basis of the resolution of the neighboring isomer peaks, different parameters (i.e., pH and concentration of buffer, surfactant concentrations, length of the injected micellar plug, organic modifier, and applied separation voltage) were optimized to achieve a satisfactory PF-MEKC separation. Under optimum conditions, the separation of 13 cytokinin standards was accomplished within 25 min. MS/MS with multiple reaction monitoring detection was carried out to obtain sufficient selectivity. PF-MEKC-MS/MS allowed for the direct identification and confirmation of the cytokinins present in banana (Musa spp.) pulp sample after extraction and purification. Finally, trans-zeatin riboside (ZR) and trans-zeatin (Z) were unambiguously identified in banana pulp. It is anticipated that the current PF-MEKC-MS method can be applied to analyze cytokinins in a wide range of biological samples.