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Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Isatis tinctoria L. For the efficient production of flavonoids and evaluation of antioxidant activities.

Gai QY, Jiao J, Luo M, Wei ZF, Zu YG, Ma W, Fu YJ - PLoS ONE (2015)

Bottom Line: I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes.Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW).To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, 150040, PR China; Collaborative Innovation Center for Development and Utilization of Forest Resources, Harbin, 150040, PR China.

ABSTRACT
In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC₅₀ values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.

No MeSH data available.


Related in: MedlinePlus

Selection of high-productive ITHRL.(A) Biomass production and (B) FL accumulation of eight selected ITHRL (I-VIII) after 3 weeks of cultivation in MS/2 liquid medium (initial pH 5.8, inoculum size 0.5%, culture temperature 25°C and sucrose concentration 3%). (C) Cultivation of ITHRL V on MS/2 solid medium and (D) its prolific growth in MS/2 liquid medium. Mean ± SD values not sharing the same lowercase letters are significantly different (P < 0.05).
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pone.0119022.g001: Selection of high-productive ITHRL.(A) Biomass production and (B) FL accumulation of eight selected ITHRL (I-VIII) after 3 weeks of cultivation in MS/2 liquid medium (initial pH 5.8, inoculum size 0.5%, culture temperature 25°C and sucrose concentration 3%). (C) Cultivation of ITHRL V on MS/2 solid medium and (D) its prolific growth in MS/2 liquid medium. Mean ± SD values not sharing the same lowercase letters are significantly different (P < 0.05).

Mentions: Based on results of preliminary experiments (data not shown), the petiole sections of I. tinctoria were provided as the potential donor explants for the genetic transformation with A. rhizogenes strain LBA9402. The successful transformation was indicated by the direct emergence of roots, formation of callus or gall-like structures from the wounded sites of explants. The optimal transformation rate (76.67%) was obtained when 3 week-old petiole explants were co-cultured for 2 days with the supplementation of 125 μM acetosyringone and 1.5 mM arginine. The transformed roots would emerge at the wounded sites of explants after 3 weeks of cultivation. Subsequently, the independently transformed hairy root lines were excised and sub-cultured on MS/2 solid medium with antibiotics to eliminate bacteria. As presented in Fig. 1C, the established I. tinctoria hairy roots exhibited the typical morphological characteristics with vigorous growth on phytohormone-free medium, lack of geotropism and extensive lateral branching.


Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Isatis tinctoria L. For the efficient production of flavonoids and evaluation of antioxidant activities.

Gai QY, Jiao J, Luo M, Wei ZF, Zu YG, Ma W, Fu YJ - PLoS ONE (2015)

Selection of high-productive ITHRL.(A) Biomass production and (B) FL accumulation of eight selected ITHRL (I-VIII) after 3 weeks of cultivation in MS/2 liquid medium (initial pH 5.8, inoculum size 0.5%, culture temperature 25°C and sucrose concentration 3%). (C) Cultivation of ITHRL V on MS/2 solid medium and (D) its prolific growth in MS/2 liquid medium. Mean ± SD values not sharing the same lowercase letters are significantly different (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4364778&req=5

pone.0119022.g001: Selection of high-productive ITHRL.(A) Biomass production and (B) FL accumulation of eight selected ITHRL (I-VIII) after 3 weeks of cultivation in MS/2 liquid medium (initial pH 5.8, inoculum size 0.5%, culture temperature 25°C and sucrose concentration 3%). (C) Cultivation of ITHRL V on MS/2 solid medium and (D) its prolific growth in MS/2 liquid medium. Mean ± SD values not sharing the same lowercase letters are significantly different (P < 0.05).
Mentions: Based on results of preliminary experiments (data not shown), the petiole sections of I. tinctoria were provided as the potential donor explants for the genetic transformation with A. rhizogenes strain LBA9402. The successful transformation was indicated by the direct emergence of roots, formation of callus or gall-like structures from the wounded sites of explants. The optimal transformation rate (76.67%) was obtained when 3 week-old petiole explants were co-cultured for 2 days with the supplementation of 125 μM acetosyringone and 1.5 mM arginine. The transformed roots would emerge at the wounded sites of explants after 3 weeks of cultivation. Subsequently, the independently transformed hairy root lines were excised and sub-cultured on MS/2 solid medium with antibiotics to eliminate bacteria. As presented in Fig. 1C, the established I. tinctoria hairy roots exhibited the typical morphological characteristics with vigorous growth on phytohormone-free medium, lack of geotropism and extensive lateral branching.

Bottom Line: I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes.Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW).To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, 150040, PR China; Collaborative Innovation Center for Development and Utilization of Forest Resources, Harbin, 150040, PR China.

ABSTRACT
In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC₅₀ values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.

No MeSH data available.


Related in: MedlinePlus