In vitro-derived plants grown from low nitrate medium produced quality tubers in Decalepis hamiltonii Wight & Arn.

In the present study, influence of nitrate concentration on in vitro rooting and its ex vitro hardened tubers were investigated in D. hamiltonii. In vitro multiple shoots established on the MS medium were used as explants for this study and concentration of indole-3-butyric acid (1.23 µM) required for root initiation was determined. The effect of different nitrate concentrations in MS medium for in vitro rooting was investigated and positive (» and ½ strength) influence was observed on 14 days inoculation. The in vitro rooted plants (IVP) showed 80% survival upon hardening and exhibited similar growth pattern to seedling plants. The 1-year grew ex vitro hardened plants were evaluated for their tuber quality with reference to their yield and flavor metabolite 2-hydroxy-4-methoxy benzaldehyde (2H4MB) content. The IVP grown from » nitrate strength media produced 155 ± 4.85 g FW of tuber biomass which is observed to be higher than full nitrate strength IVP (112 ± 2.52 g) which are grown under similar conditions in the greenhouse. The flavor metabolite content, total phenolics, total flavonoids, and antioxidant potential of these IVP tubers were evaluated. Upregulation of flavor biosynthetic pathway genes DhPAL, DhC4H and DhCoMT were observed in tubers of potted plants that developed from low nitrate strength culture media. In this study, superiority of tissue cultured plants was evident, wherein in vitro plants developed in low nitrate strength medium and acclimatized could produce a higher yield in tuber biomass and maintain relative content of flavor compounds in this endangered plant.

Proteomic approach to identify the differentially abundant proteins during flavour development in tuberous roots of Decalepis hamiltonii Wight & Arn.

2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB) is a structural isomer of vanillin produced in the tuberous roots of D. hamiltonii. Both vanillin and 2H4MB share the common phenylpropanoid pathway for their synthesis. Unlike vanillin, in which the biosynthetic pathway was well elucidated in V. planifolia, the 2H4MB biosynthetic pathway is not known in any of its plant sources. To find the key enzymes/proteins that promote 2H4MB biosynthesis, a comparative proteomic approach was adapted. In this case, two developmental stages of tuberous roots of D. hamiltonii were selected, where the flavour content was highly variable. The flavour content in the two stages was estimated using quantitative HPLC. The flavour content in the first and second stages of tuber development was 160 and 510 µgg-1, respectively. Two-dimensional electrophoresis (2-DE) was performed for these two stages of tubers; this was followed by PDquest analysis. A total of 180 protein spots were differentially abundant of which 57 spots were selected and subjected to MALDI-TOF-TOF analysis. The largest percentage of identified proteins was involved in stress and defence (27.9%), followed by proteins related to bioenergy and metabolism (23.2%), Cellular homeostasis proteins (18.6%), signaling proteins (11.6%), Plant growth and development proteins (9.3%). Holistically, we found the upregulation of methyltransferase, cell division responsive proteins, plant growth and development proteins which directly relate to flavour development and maturation. Similarly, stress-responsive and signaling proteins, vacuole proteins and ATPases were down-regulated with an increase in flavour content. In this study, we could not identify the specific 2H4MB metabolic pathway proteins, however, we could be able to study the changes in physiological and primary metabolic proteins with 2H4MB accumulation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02714-x.

Biochemical and molecular insights of PGPR application for the augmentation of carotenoids, tocopherols, and folate in the foliage of Moringa oleifera.

Plant Growth Promoting Rhizobacteria (PGPR) were utilized to contemplate their impact on the foliage of Moringa oleifera and examined for changes in tocopherols, chlorophyll, carotenoids, and folate in the sixth week. Among the eight treatments, Bacillus subtilis GB03, B. pumilus SE34, B. pumilus T4, and Pseudomonas fluorescens UOM14 improved α-tocopherol (10-14 fold) and ß-carotene (1-1.40 fold) altogether significantly (P ≤ 0.05). The most significant improvement in folate content was apparent for B. subtilis IN937B (5.47 fold) trailed by B. pumilus SE34 (5.05 fold) and B. pumilus T4 (5.12 fold) treatments. P. fluorescens UOM14 indicated remarkable improvement in Chl a (0.39 fold) and Chl b (0.44 fold) content. Organisms showing a significant increase for the analyzed molecules in individual treatment were blended in different combinations and were used for the next set of treatments. Of all the three combinations, Combination 2 (COM2-B. pumilus SE34 + B. pumilus T4 + B. pumilus INR7) showed the maximum increase in α-tocopherol (8.46 fold) and γ-tocopherol (8.45 fold), followed by Combination 3 (COM3-B. pumilus SE34 + B. pumilus T4 + P. fluorescens UOM14) (5.93 and 3.65 fold). On the whole COM2 containing different strains of B. pumilus was found to enhance the targeted metabolites in foliage significantly. Real-time PCR studies were conducted for the biochemical pathway genes of the targeted molecules, including, γ-tocopherol methyltransferase (γ-TMT), phytoene synthase (PSY), phytoene desaturase (PDS), lycopene ß cyclase (LBC) and dihydrofolate reductase thymidylate synthase (DHFR-TS). All the selected genes exhibited an up-regulation compared to control, similar to the biochemical output. Our investigation provides the strong evidence that PGPR can be viably utilized in combination to enhance the quality of the food crops.

Expression profile of phenylpropanoid pathway genes in Decalepis hamiltonii tuberous roots during flavour development.

To explore the transcriptional control of phenylpropanoid pathway (PPP) involved in vanillin flavour metabolites production in tuberous roots of Decalepis hamiltonii, four PPP key genes expressed during the tuber development were identified and their mRNA expression profiles were evaluated using quantitative real-time PCR. Flavour metabolite quantification by HPLC analysis confirmed 10, 170 and 500 µg/g 2-hydroxy-4-methoxy benzaldehyde and 4, 20 and 40 µg/g vanillin in first- (3-month-old plant), second- (18-month-old plant) and third-stage tubers (60-month-old matured plant), respectively. The expression of all four genes phenylalanine ammonia lyase (DhPAL), cinnamate-4-hydroxylase (DhC4H), caffeic acid-O-methyltransferase (DhCOMT) and vanillin synthase (DhVAN) increased with flavour development from first stage to second stage. A decrease in expression from 1.9-folds to 0.1-folds and 19.2-folds to 5.2-folds for DhCOMT and DhVAN was recorded for second stage to third stage, respectively. However, a gradual increase in expression of DhPAL (up to 26.4-folds) and a constant expression pattern for DhC4H (up to 7.1-folds) was evident from second stage to third stage of flavour development. The decrease in the expression levels of DhCOMT and DhVAN in third stage shows that the second-stage tubers are more transcriptionally active towards flavour biosynthesis.

Medium composition potentially regulates the anthocyanin production from suspension culture of Daucus carota.

In the present study, an effort has been made to optimize various culture conditions for enhanced production of anthocyanin. Nutrient content of MS medium (ammonium to potassium nitrate ratio and phosphate concentration) had a profound influence on the cell biomass and anthocyanin accumulation in cell suspension cultures of Daucus carota. Suspension cultures were carried out in shake flasks for 18 days and examined for cell growth, anthocyanin synthesis, anthocyanin yield and development of pigmented cells in relation to the uptake of total sugar, extracellular phosphate, nitrate and ammonia. The addition of NH4NO3 to KNO3 ratio (20.0 mM: 37.6 mM) in the suspension culture media resulted in a 2.85-fold increase in anthocyanin content at day 3. Similarly, a lower concentration of KH2PO4 (0.45 mM) in the MS medium resulted in 1.63-fold increase in anthocyanin content at day 9. The total sugar uptake was closely associated with a significant increase in anthocyanin accumulation. Total sugar and nitrate were consumed until 9-12 days, while ammonia and phosphate were completely consumed within 3 days after inoculation. After 9 days, cell lysis was observed and resulted in the leakage of intracellular substances. These observations suggest that anthocyanin was synthesized only by viable pigmented cells and degraded rapidly after cell death and lysis. This study signifies the utility of D. carota suspension culture for further up-scaling studies of anthocyanin.

Enhanced production of vanillin flavour metabolites by precursor feeding in cell suspension cultures of Decalepis hamiltonii Wight & Arn., in shake flask culture.

The flavour rich tuberous roots of Decalepis hamiltonii are known for its edible and medicinal use and have become endangered due to commercial over-exploitation. Besides 2-Hydroxy-4-methoxy benzaldehyde (2H4MB), other flavour metabolites in tuberous roots include vanillin, 4-Methoxy Cinnamic acid derivatives, aromatic alcohols etc. So far, there are no reports on the pathway of 2H4MB biosynthesis nor there is an organized work on biotransformation using normal and cell suspension cultures for obtaining these metabolites using precursors. The main aim of the study is to develop a method for enhanced production of flavour attributing metabolites through ferulic acid (FA) feeding to the D. hamiltonii callus culture medium. Biomass of D. hamiltonii cell suspension cultures was maximum (200.38 ± 1.56 g/l) by 4th week. Maximum production of 2H4MB was recorded on 4th week (0.08 ± 0.01 mg/100 g dry weight) as quantified by HPLC. Addition of 0.1-1.5 mM ferulic acid as precursor in the culture medium showed significant (p < 0.001) effect on suspension cultures biomass and respective phenylpropanoid metabolites content and 2H4MB accumulation. The maximum accumulation of vanillin, 2H4MB, vanillic acid, ferulic acid were of 0.1 ± 0.02 mg/100 g, 0.44 ± 0.01 mg/100 g, 0.52 ± 0.04 mg/100 g, 0.18 ± 0.02 mg/100 g DW respectively in 4 weeks of cultured cells supplemented with 1 mM ferulic acid as a precursor. The results indicate that, substantial increase in the levels of flavour metabolites in D. hamiltonii callus suspension culture was achieved. This would be having implications in biosynthesis of respective vanilla flavour attributing metabolites at very high levels for their large scale production.

Biochemical characterization of a key step involved in 2H4MB production in Decalepis hamiltonii.

Decalepis hamiltonii is widely known for its flavour molecule 2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB), a structural isomer of vanillin. As the biosynthetic pathway of 2H4MB is not known, we hypothesised 2H4MB origins could be from phenylpropanoid pathway (PPP). Accordingly, a study was conducted using PPP inhibitors (viz. piperonylic acid, MDCA and propanil) against in vitro root cultures of D. hamiltonii to find the branch of PPP which catalyses the 2H4MB formation. HPLC analysis was carried out to quantify 2H4MB levels in control and respective inhibitor treated root cultures in vitro. The results obtained revealed that piperonylic acid did not inhibit 2H4MB biosynthesis in the given period, whereas MDCA and propanil had the marked inhibitory effect. The inhibitory effect was evident with 13.2, 33.6 and 37.9% decrease in 2H4MB levels at 50, 100 and 150mM concentration of MDCA respectively in comparison with control roots. Similarly, the inhibitory effect of propanil on 2H4MB biosynthesis was obvious with 23.7, 49.5 and 57.9% decrease in 2H4MB levels at 50, 100 and 150µM concentration of inhibitor respectively when compared with control roots. Propanil showed a greater slow down effect on 2H4MB biosynthesis compared to MDCA. Incorporation of 0.1, 0.5 and 1.0mM ferulic acid as a precursor to in vitro root cultures of D. hamiltonii showed an increase in 2H4MB levels at the rate of 3.1, 107 and 94.1% respectively as quantified by HPLC analysis. However, ferulic acid in conjunction with propanil did not show any increase in 2H4MB levels. This clearly explains that ferulic acid is channelled through the 4-CL (4-coumarate CoA ligase) enzyme, where it would be converted to feruloyl-CoA and could be further converted to 2H4MB in D. hamiltonii.

Impacts of biotic and abiotic stress on major quality attributing metabolites of coffee beans.

Biotic stress factors such as Rhizopus oligosporus and Aspergillus niger mycelial extracts and abiotic elements methyljasmonate (MJ) and salicylic acid (SA), when administered through floral spray to Coffea canephora, showed significant influence on major bioactive metabolites of beans. Up to 42% caffeine, 39% theobromine and 46% trigonelline, along with 32% cafestol and kahweol content elevation was evident under respective elicitor treatments. Over all, the surge in respective metabolites depends on elicitor stress type and concentration. Abiotic factors MJ and SA were found to be efficient at 1 to 5 microM concentration in augmenting all the metabolites, compared to R. oligosporus and A. niger spray at 0.5-2.0% wherein the response was moderate as compared to abiotic stress, however significant compared to control. Though this elevation in caffeine, theobromine, cafestol and kahweol is not warranted from quality point of view, increase in trigonelline improves coffee quality. Besides increase in metabolites, stress mediated augmentation of bioactive compounds in coffee has a wide scope for studying gene expression pattern.

Culture Conditions for Production of Biomass, Adenosine, and Cordycepin from Cordyceps sinensis CS1197: Optimization by Desirability Function Method.

BACKGROUND: Cordyceps sinensis (CS) is a traditional Chinese medicine contains potent active metabolites such as nucleosides and polysaccharides. The submerged cultivation technique is studied for the large scale production of CS for biomass and metabolites production. OBJECTIVE: To optimize culture conditions for large-scale production of CS1197 biomass and metabolites production. MATERIALS AND METHODS: The CS1197 strain of CS was isolated from dead larvae of natural CS and the authenticity was assured by the presence of two major markers adenosine and cordycepin by high performance liquid chromatography and mass spectrometry. A three-level Box-Behnken design was employed to optimize process parameters culturing temperature, pH, and inoculum volume for the biomass yield, adenosine and cordycepin. The experimental results were regressed to a second-order polynomial equation by a multiple regression analysis for the prediction of biomass yield, adenosine and cordycepin production. Multiple responses were optimized based on desirability function method. RESULTS: The desirability function suggested the process conditions temperature 28°C, pH 7 and inoculum volume 10% for optimal production of nutraceuticals in the biomass. The water extracts from dried CS1197 mycelia showed good inhibition for 2 diphenyl-1-picrylhydrazyl and 2,2-azinobis-(3-ethyl-benzo-thiazoline-6-sulfonic acid-free radicals. CONCLUSION: The result suggests that response surface methodology-desirability function coupled approach can successfully optimize the culture conditions for CS1197. SUMMARY: Authentication of CS1197 strain by the presence of adenosine and cordycepin and culturing period was determined to be for 14 daysContent of nucleosides in natural CS was found higher than in cultured CS1197 myceliumBox-Behnken design to optimize critical cultural conditions: temperature, pH and inoculum volumeWater extract showed better antioxidant activity proving credible source of natural antioxidants.

AgNO3: a potential regulator of ethylene activity and plant growth modulator

The aim of this review is to critically analyze the role of silver nitrate (AgNO3) in modulating plant growth and development. In recent years, basic studies on ethylene regulation opened new vistas for applied research in the area of micro-propagation, somatic embryogenesis, in vitro flowering, growth promotion, fruit ripening, and sex expression. Silver nitrate has proved to be a very potent inhibitor of ethylene action and is widely used in plant tissue culture. Few properties of silver nitrate such as easy availability, solubility in water, specificity and stability make it very useful for various applications in exploiting plant growth regulation and morphogenesis in vivo and in vitro. Silver ion mediated responses seem to be involved in polyamines, ethylene- and calcium- mediated pathways, and play a crucial role in regulating physiological process including morphogenesis. The molecular basis for regulation of morphogenesis under the influence of silver nitrate is completely lacking. This review compiles published reports of silver nitrate-mediated in vitro and in vivo studies and focuses on fundamental and applied aspects of plant growth modulation under the influence of silver nitrate.