Different Strains Of Agrobacterium Rhizogenes Research Articles

Enhanced production of bioactive plumbagin in hairy root cultures and adventitious root cultures of Plumbago zeylanica L. by a novel apocarotenoid elicitor, α-ionone

Plumbagin, a medicinally important secondary metabolite is produced in the roots of Plumbago zeylanica Linn. Natural harvest of plumbagin from roots is a destructive process that destroys the entire plant. The aim of this study was the in vitro production of plumbagin at high concentrations. For this purpose, hairy root cultures (HRCs) and adventitious root cultures (ARCs) were induced from P. zeylanica leaf explants. Additionally, first-time α-ionone was used as an elicitor to trigger plumbagin biosynthesis. HRCs were developed using the two different strains of Agrobacterium rhizogenes (LBA1334, and R1000). R1000 strain showed the highest HR induction efficiency. ARCs were induced from the leaf explants growing in Murashige and Skoog (MS) solid media supplemented with 1 mg/L indole-3-butyric acid. Newly emerged adventitious roots were used to develop root suspension cultures in a liquid MS medium. Plumbagin content was determined by HPLC/DAD. Both HRCs and ARCs showed fast growth kinetics. The greatest increase in the production of plumbagin was achieved with 10 µM α-ionone. As compared to untreated controls, the optimum α-ionone dose resulted in a 3.6-fold and 3.1-fold increase in plumbagin in HRCs, and ARCs, respectively. These results support that α-ionone elicitor-treated HRCs and ARCs are promising technology for improved production of plumbagin to meet commercial demand.

Harnessing the Potential of Roots of Traditional Power Plant: Ocimum.

Genus Ocimum of Labiatae is well known in all traditional medicinal systems like Ayurveda, Unani, Siddha, and Homeopathy. The pharmaceutical activities of different species of Ocimum attributed to all plant parts. Roots are the most significant vital organ of the plant, as they absorb water and nutrients from soil and transport to aerial parts of the plants. Roots of Ocimum were found helpful with free-radical scavenging activity to improve physical and mental strength as well as to treat diabetes, malaria, and liver problems. Antibacterial activity of Ocimum roots and its main component, rosmarinic acid, is very beneficial to protect against several human pathogens, including bacteria and viruses. Being so important in every way, roots of Ocimum need healthy rhizosphere. Bacteria, fungi, nematodes, types of soil, fungicide, pesticides, salt, radioactive elements, as well as heavy metal contaminations, affect roots and overall growth of Ocimum in positive or negative ways. Each component of rhizosphere (natural, treatment or contamination) affects the roots, which highlights current ecological scenario to discover biosafe and more productive approaches. For such prestigious organ of Ocimum, development of in vitro root cultures and hairy root cultures assists to reduce the efforts and timing of the traditional cultivation process along with elimination of negative factors in rhizosphere. Different strains of Agrobacterium rhizogenes, various media compositions, as well as discrete treatments, like elicitors, on nonidentical species or cultivars of Ocimum boost the root induction, biomass, and accumulation of phytoceuticals differently. Hairy roots and in vitro roots of Ocimum accumulate higher quantity of therapeutic metabolites. These metabolites include several phenolics (like rosmarinic acid, 3-hydroxybenzoic acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, vanillic acid, chicoric acid, and lithospermic acid), triterpenes (such as betulinic acid, 3-epimaslinic acid, alphitolic acid, euscaphic acids, oleanolic acid, and ursolic acid) as well as flavonoids (flavones, flavonols, and dihydroflavonols). This review highlights pharmaceutical applications of Ocimum roots, a great deal of rhizosphere components and in vitro culturing techniques to enhance biomass as well as chief phytoceuticals.

Iron and zinc oxide nanoparticles: An efficient elicitor to enhance trigonelline alkaloid production in hairy roots of fenugreek

Fenugreek (Trigonella foenum-graecum L.) is a rich source of important medicinal metabolites. This plant belongs to the Fabaceae family that grows anywhere around the world. It is extensively used in medicine and as a food additive. Several secondary metabolites are detected in fenugreek, especially a valuable alkaloid called trigonelline. Enhancing the production of secondary metabolites is possible through the application of modern biotechnological techniques such as culturing hairy roots. In the present study, different strains of Agrobacterium rhizogenes (ATCC11325, ATCC15834, A4, A7, A13, and K599) were used for hairy root induction from fenugreek cotyledon explants. Then, the effect of various levels (x, 2x, and 4x concentrations of B5 medium) of iron oxide and zinc oxide nanoparticles were evaluated on growth, total phenolic, flavonoids, and trigonelline content of hairy roots. The growth curve analysis revealed an increase in the fresh and dry weight of treated hairy roots, as compared with the control. The highest levels of fresh (1.49 g) and dry weight (1.23 g) were obtained in hairy roots which were induced by the ATCC11325 strain and were treated with Zn (2x). The results indicated that using Zn nanoparticles on hairy roots can lead to higher levels of trigonelline, compared with the control group (i.e. transformed hairy roots without NPs).

Agrobacterium rhizogenes mediated transformation of Ficus carica L. for the efficient production of secondary metabolites.

Ficus carica L., an ancient source of food and medicines, is rich in valuable nutritional and secondary compounds with antioxidant, antimicrobial, and anticancer effects. The present study is the first attempt to examine hairy root (HR) induction of F. carica (Sabz and Siah) by inoculating the 3-week-old shoots and leaves with different strains of Agrobacterium rhizogenes and also to investigate methyl jasmonate (MeJA) elicitation of HRs to produce a fast and high-yield production method for secondary metabolites. The maximum transformation rate (100%) was achieved by inoculating the shoots with Agrobacterium rhizogenes strain A7. Siah HRs elicited with 100 and 200 μmol L-1 MeJA and Sabz HRs with 100 μmol L-1 MeJA showed the highest total phenolic content. The highest flavonoid content was 3.935 mg QE g-1 DW in Siah HRs treated with 200 μmol L-1 MeJA and 2.762 mg QE g-1 DW in Sabz HRs treated with 300 μmol L-1 MeJA. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and ferric reducing antioxidant power (FRAP) value of HRs were affected by MeJA treatments. Methyl jasmonate elicitation also significantly enhanced the content of six phenolic acids (gallic acid, caffeic acid, chlorogenic acid, coumaric acid, rosmarinic acid, and cinnamic acid) and three flavonoids (rutin, quercetin, and apigenin). Thymol, a monoterpene phenol, was the main HR compound detected in gas chromatography mass spectrometry (GC-MS) analysis of the essential oils. Induction of HRs and elicitation of F. carica HRs by MeJA resulted in a significant increase in the production of important phenolic compounds and a significant increase in antioxidant capacity. © 2020 Society of Chemical Industry.

Evaluation of influence of different strains of Agrobacterium rhizogenes on efficiency of hairy root induction in Rauwolfia serpentina

An improved protocol was established for hairy root induction in Rauwolfia serpentina using two strains of Agrobacterium rhizogenes MTCC 532 and 2364. Hairy root induction efficiency was standardized based on infection time, co-cultivation period, OD600 and acetosyringone concentration. Higher transformation efficiency was established using MTCC 532 (31%) and MTCC 2364 (24%) with 30 min infection time, 72 h co-cultivation period and 0.6 OD600. Transformation efficiency was further enhanced to 55 % with 125 μM acetosyringone. MTCC 532 was proven a better strain over the MTCC 2364 in all the tested factors. Putative transgenic hairy roots were confirmed by polymerase chain reaction using rolA specific primers. Biomass of randomly selected 5 hairy root lines was also significantly enhanced. No significant difference of growth was recorded among the lines except line no 4. In the present study, an enhanced system for Agrobacterium rhizogenesmediated hairy root culture was established which offer an effective means to attain improved transformation efficiency and ultimately beneficial for the industrial scale in vitro production of the secondary metabolite.

The novel paclitaxel-producing system: establishment of Corylus avellana L. hairy root culture

Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA4, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g−1 (DW)) in hairy root extracts.

Hairy roots of pea mutants with a modified morph leaf type

In the present study, some pea mutants rich in protein with a modified morph leaf type with af and tl genes were genetically transformed using different strains of Agrobacterium rhizogenes A-4, A-48, 15834. Even though a higher total protein content was obtained with the A-48 strain, maximum of the probable transformants were obtained with the A-4 strain but of transient character. The content of the total protein in hairy roots cultures, depending on the composition of the nutrient media and the growth phase of the culture, has been refined.

High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Cichorium intybus L. via removing macronutrients

Transformed hairy roots technology provides new opportunities for mass production of pharmaceutical metabolites. Hairy roots culture has been considered as an alternative method for producing medicinal biomolecules in plants. Chicory (Cichorium intybus L.) is a medicinal herb from Asteraceae family. It contains many important metabolites including chicoric acid, inulin, scoline, coumarin, and flavonoids. In this study, for the first time, a reliable gene transfer system via modifying co-cultured medium using different strains of Agrobacterium rhizogenes and explants were established for C. intybus. Different co-culture media were used for this purpose, including MS basal medium and MS medium without either of KNO3, NH4NO3, KH2PO4, CaCl2, and MgSO4. The results showed increases in hairy roots induction percentage, biomass production, and phenolic content of the hairy roots. Maximum hairy root induction percentage obtained by using A4 strain when a KNO3-free MS medium was used. High frequency of hairy root induction percentage with ATCC11325 and ATCC15834 strains was achieved when MS medium had KH2PO4 removed. In all of the experiments, very low hairy root induction was observed when NH4NO3 was removed from co-culture MS medium. Molecular confirmation of transgenic hairy roots was done with PCR using gene-specific primers for rolB gene. Total phenols, flavonoids, anthocyanins and chicoric acid contents of hairy root clones and the control (untransformed) roots were also determined. The results indicated significant increases in total metabolites content of hairy root clones induced by A4, ATCC11325 and ATCC15834 strains with some macronatrient–free co-culture MS media and also directly-regenerated shoots from hairy roots were spontaneous without using any plant growth regulators.

Enhanced production of pharmaceutically important isoflavones from hairy root rhizoclones of Trifolium pratense L.

These studies report the development of an efficient technique for large-scale cultivation of fast-growing hairy root culture systems for production of bioactive isoflavones. Trifolium pratense L. is an important source of pharmaceutically important isoflavones with immense health care applications. Trifolium pratense was transformed using different strains of Agrobacterium rhizogenes for hairy root induction and establishment of hairy root rhizoclones. Selected fast-growing rhizoclones of T. pratense were evaluated for their growth and isoflavone production. This study is the first report of stable production of isoflavones through successive culture passages from transformed hairy-root rhizoclones of T. pratense. One of the fast-growing hairy-root rhizoclones 2364A displayed significantly higher accumulation of all four pharmaceutically important isoflavones, 8.56 mg (gdw)−1 of daidzein, 2.45 mg (gdw)−1 of genistein, 15.23 mg (gdw)−1 of formononetin, and 1.10 mg (gdw)−1 of biochanin A, compared to other rhizoclones.

Optimizing culture conditions for establishment of hairy root culture of Semecarpus anacardium L.

Semecarpus anacardium L. is a tree species which produces secondary metabolites of medicinal importance. Roots of the plant have been traditionally used in folk medicines. Different strains of Agrobacterium rhizogenes (A4, ATCC15834 and LBA 9402) were used for induction of hairy roots in in vitro grown tissues of the plant. Hairy root initiation was observed after 25–30 days of infection. Optimum transformation frequency of 61% was achieved on leaf explants with ATCC15834 strain. Infection time of 30 min resulted in greater transformation frequency compared to 10 and 20 min, respectively. The hairy roots cultured in growth regulator-free semi-solid woody plant medium differentiated into callus. Whole shoots infected with ATCC 15834 were found to produce more transformants upon co-cultivation for 4 (65%) and 5 (67%) days. Induction of hairy roots in stem explants infected with ATCC 15834 was lower (52%) compared to leaves (62%) after 4 days of co-cultivation. In A4 and LBA9402 strains transformation efficiency was 49 ± 2.8% and 36 ± 5.7% in shoots after 4 days of co-cultivation. Transformation frequency was higher in ATCC15834 strain, irrespective of explants. The hairy roots of S. anacardium elongated slowly upon transfer to half-strength liquid medium. After 3–4 passages in liquid medium slender hairy roots started differentiating which were separated from the original explants. Visible growth of the roots was observed in hormone-free liquid medium after 2–3 months of culturing. Polymerase chain reaction with gene-specific primers from rol A, B and C genes confirms the positive transformation events.

Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNTs chromatography

Tissue-type plasminogen activator (tPA) is a serine protease that plays a crucial role in the fibrinolytic system. We increased the activity of tPA by splicing the active site of dodder-cuscutain gene to human tPA. The chimeric cDNA of tPA was constructed by Splicing by Overlap Extension Polymerase Chain Reaction (SOEing-PCR) method and transferred to the hairy roots of tobacco using different strains of Agrobacterium rhizogenes. Chimeric-tPA was purified by lysine-sepharose chromatography and specific aptamers were designed using SELEX method. Multi wall carbon nanotubes were functionalized with selected aptamers, packed in a column, and used for purification. The results demonstrated that selected aptamer having KD values of 0.320 nM and IC50 of 28.9 nM possessed good affinity to tPA, and the chimeric-tPA was properly purified by aptamer-chromatography. Hairy roots expressing chimeric-tPA and normal-tPA produced 900 and 450 ngmg−1 of total protein, respectively. The activities of chimeric–tPA and normal–tPA were 90 and 60 IUml−1, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.

Hairy roots culture as a source of valuable biopharmaceuticals

Plants have been exploited as a source of medicinal substances for years. Nowadays, achievements of modern science, including molecular biotechnology, allow their huge potential to be utilized. They have become a promising platform for the production of valuable compounds such as biopharmaceuticals. Among the various plant systems used for this purpose, hairy root cultures are also applied for the production of recombinant proteins and secondary metabolites. For this purpose plant cells of selected species are genetically transformed using different strains of Agrobacterium rhizogenes carrying the desired genes. The next steps of this process include stable and efficient expression of these genes. Hairy root cultures exhibit a number of features which make them attractive compared to various pro- and eukaryotic cell systems including other plant models. Their main advantages are: relatively low production costs, ease of scale-up, production of compounds typical for eukaryotic cells with post-translational modifications, biological safety, and in many cases there is no need for complex purification techniques of the final product. Several compounds that are successfully obtained using this production strategy are valuable pharmaceuticals. This group includes selected cytokines, vaccine antigens and antibodies.

Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC.

An efficient protocol for hairy root induction in Berberis aristata DC. was established using two different strains of Agrobacterium rhizogenes, MTCC 532 and 2364 from IMTECH (Institute of Microbial Technology), Chandigarh, India. The strain 532 was more effective than strain 2364 in hairy root induction and in vitro grown callus (61.11 ± 1.60 % transformation frequency) was found to be suitable explant in comparison to leaves (42.59 ± 0.92 % transformation frequency) and nodal segments (34.25 ± 0.92 % transformation frequency) of in vitro grown microshoots for hairy root induction. The presence of rol A and rol B genes during amplification confirmed the transgenic nature of hairy roots and transformed callus. Transformation frequency of callus was further enhanced (from 61.11 ± 1.60 % to 72.22 ± 1.60 %; when infection time was 1 h) by using acetosyringone (100 µM) during co-cultivation period (48 h) on semisolid MS (Murashige and Skoog) medium. In conclusion, this study describes the protocol for hairy root induction which could further be useful for the production of berberin and may reduce the overharvesting of this endangered species from its natural habitat.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1222-1) contains supplementary material, which is available to authorized users.

Establishment of Persicaria minor hairy roots and analysis of secreted β-caryophyllene in medium broth

A method for hairy root induction of Persicaria minor, was developed and investigated, and a qualitative evaluation of the sesquiterpenes in the culture medium was performed. The transgenic status was confirmed by PCR analysis using rolB and virD specific primers. The efficiency with which four different strains of Agrobacterium rhizogenes (A4, ATCC43056, ATCC15834 and ATCC13333) and different concentrations of acetosyringone induced hairy roots from in vitro leaf segments of P. minor was evaluated. Strains A4 and ATCC13333 were able to induce hairy root in an average of 6 days of co-cultivation and exhibited significantly higher transformation efficiencies, 20 and 21.3 %, respectively, compared to the other strains in the presence of 100 µM acetosyringone. Half-strength Murashige–Skoog basal medium supplemented with Gamborg vitamins supported the greatest increase (35-fold) of growth index of hairy roots relative to the initial weight after 8 weeks of culture. GC–MS results showed that β-caryophyllene was the main sesquiterpenes detected and was quantified with standards, with total peak areas of 7.934. Results suggested that hairy roots secreted sesquiterpenes into the medium while preserving viable hairy roots as a source of specialized metabolites.

English

Carrot hairy root induction by two different strains of Agrobacterium rhizogenes- MTCC- 532 and MTCC- 2364 grown in yeast extract mannitol agar medium (YEMA) was studied in carrot (Daucus carota). The maximum hairy root induction was observed when carrot was simmered with 48 h old culture of A. rhizogenes MTCC-532. With different concentrations of acetosyringone (AS) tested, addition of 150 µM acetosyringone was found to enhance the transformation frequency up to 75(±2.60)a and 60 (±2.08)a percentage by A. rhizogenes MTCC-532 and MTCC 2364 strains, respectively. Transformation efficiency was highly dependent on the acetosyringone concentrations, type of bacterial strains and carrot genotype. Transfer of Ri Ti-DNA was confirmed by polymerase chain reaction (PCR) analysis, the detection of ags gene in transformed carrot hairy root. A. rhizogenes transformed hairy roots had the ability to form copious lateral roots as well as a negative geotropic growth habit in a shorter period of time. The Murashige and Skoog (MS) medium was found to be the best medium for hairy root mass multiplication, which induced high root biomass production and rapid root tip elongation.  Key words: Carrot, Agrobacterium rhizogenes, acetosyringone, hairy roots, Murashige and Skoog (MS) medium, arbuscular mycorrhizae.

Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum

This article presents the abilities and efficiencies of five different strains of Agrobacterium rhizogenes (strain ATCC 31798, ATCC 43057, AR12, A4 and A13) to induce hairy roots on Solanum mammosum through genetic transformation. There is significant difference in the transformation efficiency (average number of days of hairy root induction) and transformation frequency for all strains of A. rhizogenes (P < 0.05). Both A. rhizogenes strain AR12 and A13 were able to induce hairy root at 6 days of co-cultivation, which were the fastest among those tested. However, the transformation frequencies of all five strains were below 30 %, with A. rhizogenes strain A4 and A13 showing the highest, which were 21.41 ± 10.60 % and 21.43 ± 8.13 % respectively. Subsequently, the cultures for five different hairy root lines generated by five different strains of bacteria were established. However, different hairy root lines showed different growth index under the same culture condition, with the hairy root lines induced by A. rhizogenes strain ATCC 31798 exhibited largest increase in fresh biomass at 45 days of culture under 16 h light/8 h dark photoperiod in half-strength MS medium. The slowest growing hairy root line, which was previously induced by A. rhizogenes strain A13, when cultured in optimized half-strength MS medium containing 1.5 times the standard amount of ammonium nitrate and potassium nitrate and 5 % (w/v) sucrose, had exhibited improvement in growth index, that is, the fresh biomass was almost double as compared to its initial growth in unmodified half-strength MS medium.

Agrobacterium-Mediated Transformation in Alpinia galanga (Linn.) Willd. for Enhanced Acetoxychavicol Acetate Production

Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A(4), 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A(4) transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A(4). The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.

Agrobacterium rhizogenes strains이 황금 모상근 유도와 생육에 미치는 영향

Agrobacterium rhizogenes, a gram-negative soil bacterium, is one of the most widely studied among them. A, rhizogenes can transfer T-DNA, excised from Ri (root inducing)-plasmids from the bacterial to the plant cell. It is the causal agent of ‘hairy root’ diseases in plants, and has been used for the production of hairy root cultures from a multitude of species. Five different strains of Agrobacterium rhizogenes differed in their ability to induce Scutellaria baicalensis hairy roots and also showed varying effects on the growth in hairy root cultures. A. rhizogenes R 1000 is the most effective strain for the induction (57.3%) and growth (11.9 g l?¹) in hairy root of Scutellaria baicalensis. Our results demonstrate that use of suitable strains of A. rhizogenes may allow study of the regulation of flavone biosynthesis in hairy root cultures of Scutellaria baicalensis.

Resveratrol production in hairy root culture of peanut, Arachis hypogaea L. transformed with different Agrobacterium rhizogenes strains

Five different strains of Agrobacterium rhizogenes differed in their ability to induce peanut (Arachis hypogaea L.) hairy roots and also showed varying effects on the growth and resveratrol production in hairy root cultures. A. rhizogenes R1601 is the most effective strain for the induction (75.8%), growth (7.6 g/l) and resveratrol production (1.5 mg/g) in hairy root of peanut. Our results demonstrate that the use of suitable strains of A. rhizogenes may allow study of the regulation of resveratrol biosynthesis in hairy root cultures of A. hypogaea.

Peroxidase production from hairy root cultures of red beet (Beta vulgaris)

The genetically transformed roots of red beet have been shown, for the first time, to produce very high levels of peroxidase (POD; EC 1.11.1.7) accounting for 1.21 x 10 6 Units L -1 . Of the ten clones established using different strains of Agrobacterium rhizogenes , one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods. Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L -1 ) as well as POD (ranging from 8000-9000 U g -1 FW and 1.18 x 10 6 U L -1 with a specific activity of 600 U mg -1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10 6 U L -1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of R m 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at R m 0.06, 0.16, 0.25 and one extra band of R m 0.575 in the spent medium where isozymes of R m 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.