Plant Cell Tiss Organ Cult DOI 10.1007/s11240-015-0914-0
ORIGINAL ARTICLE
Enhanced trans-resveratrol production in genetically transformed root cultures of Peanut (Arachis hypogaea L.) Mihir Halder1 • Sumita Jha1
Received: 25 August 2015 / Accepted: 14 November 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract In the present study, 30 Ri-transformed root lines of peanut (Arachis hypogaea) cv. JL-24, a popular Indian cultivar, obtained following infection with Agrobacterium rhizogenes strains LBA9402, A4 and R1000 were selected on the basis of growth index and maintained in vitro for 3 years. The root lines showed high degree of branching and rapid, plagiotropic growth on phytohormone free solid N/5 medium but were devoid of root hairs. Trans-resveratrol was isolated by preparative HPLC from Ri-transformed roots and identified by ESI–MS/MS. Strain independent variability was observed among 30 Ri-transformed root lines on the basis of lateral root density per cm (7.60 ± 0.30 to 4.5 ± 0.5), relative thickness (0.54 ± 0.07 to 1.54 ± 0.1 mm), growth index (9.16 ± 1.1 to 17.79 ± 1.35 FW basis and 10.77 ± 0.95 to 19.46 ± 1.78 DW basis) and trans-resveratrol content 0.27 ± 0.03 (root line R1000-1) to 0.969 ± 0.141 mg g DW-1 (root line RIX-19) in solid N/5 medium, which was 4.1–14 fold greater than in excised nontransformed root cultures (0.06 ± 0.01 mg g DW-1). Optimum growth and productivity in liquid culture was achieved in N/5 medium supplemented with 0.01 % activated charcoal. Root line RIX-19 showed maximum trans-resveratrol accumulation (1.21 ± 0.09 mg g DW-1) and productivity (0.37 ± 0.08 mg per flask), which was 19 fold higher than non-transformed root cultures. This optimized protocol can
Electronic supplementary material The online version of this article (doi:10.1007/s11240-015-0914-0) contains supplementary material, which is available to authorized users. & Sumita Jha
[email protected];
[email protected] 1
Department of Botany, Center of Advanced Study, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, West Bengal 700 019, India
be utilized for large scale cultivation of transformed root cultures in industrial bioreactors for mass synthesis of transresveratrol. Keywords Agrobacterium rhizogenes Arachis hypogaea Transformed root culture Rol genes Transresveratrol
Key message The present study on hairy root cultures of Peanut clearly shows that a natural transformation system and meticulous screening can be used to increase growth and achieve many fold improvement in trans-resveratrol production.
Introduction Resveratrol, a naturally occurring phytoalexin, consists of two phenolic rings linked by a styrene double bond to generate 3,5,40 -trihydroxystilbene and of the two isomeric forms of resveratrol, trans-resveratrol is sterically more stable (Trela and Waterhouse 1996), biologically more active than cis-resveratrol (King et al. 2006). Trans-resveratrol is reported to exhibit several therapeutic properties like anti-oxidant, platelet anti-aggregation, anti-inflammatory, anti-allergenic, cardio-protective and strong chemo-preventive activity against various cancers including breast, prostate and neuroblastoma (Fre´mont 2000; Chen et al. 2004; Delmas et al. 2006; Elmali et al. 2007; Hsieh and Wu 2010; Sun et al. 2010; Xu and Si 2012). Resveratrol specifically activates redox-sensing enzymes such as sirtuin-like protein deacetylases (Howitz et al. 2003), which are found in all eukaryotic organisms that monitor the cellular response to
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environmental stresses. Conditions of oxidative stress, including calorie restriction, also activate sirtuins, leading to a reduction in general metabolic activity, delayed apoptosis and increased longevity (Baur et al. 2006). Trans-resveratrol and its many derivatives (Larronde et al. 2005), are produced in a selected number of plant species such as Vitis vinifera (Liu et al. 2013), Arachis hypogaea (Sanders et al. 2000; Chen et al. 2002; Sales and Resurreccion 2014) and mulberries (Lyons et al. 2003) in low quantities. A. hypogaea L. (Peanut) is one of the important protein rich oil seed crops, native to South America, but currently grown in diverse environments throughout the world. India is the world’s second largest producer of peanut (Bishi et al. 2015). Since peanut is an important nutritional source, increasing beneficial secondary metabolites in peanut could make a significant contribution to health, particularly in develop countries. The applications of undifferentiated cell cultures for production of new pharmaceuticals have been reviewed with particular emphasis on perspectives and problems of the system (Jha et al. 2005; Weathers et al. 2010; Xu et al. 2011). The biotechnological applications of another in vitro plant system are the hairy roots, which have been reported to be potent substitute for secondary metabolite production system for root derived phytochemicals in a wide range of medicinal plants owing to the high productivity and genetic stability (Chandra 2012; Roychowdhury et al. 2013). Genetic transformation by the Ri TL-DNA of Agrobacterium rhizogenes produces roots that can be cultured in vitro (Tepfer 1984), which often show high accumulation of secondary metabolites (Jung and Tepfer 1987; Guillon et al. 2006; Georgiev et al. 2007; Tian 2015). There are few reports on A. rhizogenes mediated transformation of peanut. Medina-Bolivar et al. (2007) used a runner peanut cultivar cv. Andru II to establish hairy root cultures and reported secretion of resveratrol in culture medium. Condori et al. (2010) studied effect of elicitation on biosynthesis of stilbenoids in a hairy root line from peanut cultivar cv. Hull. This paper reports transformation of A. hypogaea cv. JL24 (a popular Indian cultivar), by different strains of A. rhizogenes and trans-resveratrol production in Ri-transformed root cultures. High yielding and fast growing Ritransformed root lines were selected for optimization of growth in liquid medium which can be used as a sustainable source of trans-resveratrol in further scale up-studies.
Materials and methods Plant material Seeds of Arachis hypogaea cv. JL-24 (a popular cultivar in India) were obtained from the International Crop Research
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Institute for the Semi-Arid Tropics (ICRISAT; Andhra Pradesh, India). The seeds were treated with 0.1 % (w/v) bavistin solution for 10 min, rinsed with water, then dipped in 70 % (v/v) ethyl alcohol for 30 s, rinsed with water for two times and finally surface sterilized with 0.1 % freshly prepared HgCl2 for 8–10 min, followed by washing three times with distilled water. Seeds were inoculated on Murashige and Skoog’s (1962) basal MS medium (MS) with 3 % (w/v) sucrose and 0.75 % (w/v) agar (Merck, India) and cultured in a growth chamber at 24 ± 1 °C and 50–60 % relative humidity under dark condition for germination. After germination seedlings were kept under 16/8 h (light/dark) photoperiod with light provided by cool-white fluorescent tubes (Philips, India) at an intensity of 48 lmol m-2 s-1. Bacterial strains Five wild type agropine strains of Agrobacterium rhizogenes, namely, LBA9402 (pRi1855; Hooykaas et al. 1977; Petit et al. 1983), A4 (pRiA4; Hooykaas et al. 1977; Cardarelli et al. 1985), R1000 (pRiA4b; Vervliet et al. 1975; Moore et al. 1979), ATCC 15834 (pRi15834; White and Nester 1980) and HRI (pRiHRI; Petit et al. 1983; Jouanin et al. 1986) were used for transformation experiments. Bacterial suspensions were prepared by inoculating a loop-full of bacteria into 10 ml of respective liquid bacterial medium (Table S1) and incubated on a gyratory shaker (Certomat) set at 180 rpm for 24–28 h at 24 ± 1 °C in dark. To improve virulence 200 lM acetosyringone (Sigma) was added to the bacterial suspensions (*1010 cells ml-1) 2 h prior to inoculation. Transformation procedure Leaflets (1.5–2.5 cm), petioles (2–2.5 cm), internodes (1–1.5 cm) and cotyledons excised from in vitro grown 20–22-day old seedling of A. hypogaea cv. JL-24 cultured on MS medium and infected with different strains of A. rhizogenes. The explants were pricked (at two sites per explants) with a 2 ml disposable sterile hypodermic needle loaded with bacterial suspension at late log phase (O.D.600nm [ 1). Liquid bacterial medium without bacterial inoculum was used for infection of explants used as a control. Infected and control explants were incubated on sterile filter paper soaked with liquid MS medium for cocultivation in the dark for 72 h. After co-cultivation period, all the explants (control and experimental) were washed thoroughly with sterile double distilled water containing 1 g l-1 ampicillin for 30 min and rinsed three times in sterile double distilled water. All the explants were then transferred and cultured on solid MS medium supplemented with 500 mg l-1 ampicillin for 28 days under 16/8 h (light/dark) photoperiod. For each experiment, 60 excised leaflets and 30 excised petioles were infected. All
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of the experiments were repeated three times. Data for putatively transformed root formation, root induction percentage per total explants (root induction frequency) and number of root induced per explant were recorded at 7 days interval for 28 days after the initial infection. Establishment and culture of induced transformed root lines
basal medium. Solid MS medium and N/5 medium supplemented with different concentrations (0.49, 2.5 and 4.9 lM) of indole 3-butyric acid (IBA) used for NT root culture. NT excised root cultures of A. hypogaea cv. JL-24 were maintained on solid N/5 medium supplemented with 2.5 lM IBA by regular sub-culturing at 28 day intervals in dark condition and designated as NTH root culture. DNA isolation and PCR analysis
Roots induced at the pricked sites of the infected explants were excised and cultured in Petri-plates (9 cm) containing 20 ml MS medium or modified MS medium (N/5 medium, Tepfer 1995) supplemented with 500 mg l-1 ampicillin in dark to establish transformed root cultures. Root segments (*3–4 cm long) with lateral branches were used as inoculum during each sub-culture. Each excised primary root was propagated as a separate root line. The amount of ampicillin in the culture medium was reduced to 250 mg l-1 after 6 months of culture with regular subculture at an interval of 28 days. To compare root growth in the two basal media (N/5 medium and MS medium), *2–3 cm long primary root segment (0.1–0.15 g fresh weight) of clones of root line RIX-33 were transferred to 9 cm Petri-plates containing the 20 ml solid medium. After 28 days of culture, the roots from each sample were harvested, washed with deionized water, blotted dry and fresh weight (FW) determined. The roots were then oven-dried at 40 °C and weighed till a constant weight was obtained and it was recorded as the dry weight (DW). Growth of roots were expressed as growth index (GI = harvest weight/inoculum weight). Five replicates were used for each experiment and each experiment was repeated three times. Maintenance of Ri-transformed root lines Each putatively transformed root line was screened for the presence of residual bacterial contamination by culturing the root segments in respective bacterial medium (Table S1) after 1 year of culture initiation. Thirty fast growing axenic Ri-transformed root lines obtained via transformation with A. rhizogenes strain LBA9402 (14 root lines), strain A4 (10 root lines) and strain R1000 (06 root lines) were selected from 150 putatively transformed root lines and axenic transformed root lines were maintained on N/5 medium for over 3 years in vitro. Excised non-transformed (NT) root culture To establish excised NT root culture, 4–5 cm long root segments were excised from 20 to 22-day old axenic seedling, grown on solid MS medium and inoculated on phytohormone unsupplemented MS basal medium and N/5
Transformation was confirmed by PCR detection of the TL-DNA and TR-DNA genes using genomic DNA extracted from 30 Ri-transformed root lines and specific primers spanning the rolA (Bonhomme et al. 2000), rolB (Wang et al. 2001), rolC (Sevo´n et al. 1997), rolD genes (Christensen et al. 2008) of TL-DNA and aux1, aux2 genes of TR-DNA (Taneja et al. 2010). Plasmid pLJ1 for TLDNA and pLJ85 for TR-DNA were used as positive control. Expected amplicon size of rolA, rolB, rolC and rolD genes were 344, 780, 545 and 402 bp respectively. To eliminate the false positive result due to A. rhizogenes contamination, the virD1 gene specific primers (Alpizar et al. 2008) were used for PCR. PCR analysis was done as described earlier (Chaudhuri et al. 2005; Roychowdhury et al. 2015). The PCR amplicons were resolved by 1.2 % (w/v) agarose gel electrophoresis with a 100 bp plus DNA ladder (Thermo Scientific, USA) and visualized by ethidium bromide staining under UV light. Documentation was done by using BioRad Gel DocTM EZ Imager. Each experiment was repeated three times. Gene expression analysis by RT-PCR The reverse transcription polymerase chain reaction (RTPCR) was performed to analyse the expression of the transgenes at the transcription level as described earlier (Majumdar et al. 2011). Thirty Ri-transformed root lines along with NT root line (used as control), were subjected to rol gene expression studies. Total RNA was isolated from 28-day old NTH root line and Ri-transformed root lines using HiPurATM Plant and Fungal RNA Miniprep Purification Kit (HIMEDIA, India) following the manufacturer protocol. Qualitative and quantitative analysis of isolated RNA was done by spectrophotometry (BioSpectrometer, eppendorf). The RT-PCR of these genes (rolA, rolB, rol C, rolD, aux1 and aux2 genes) was done following the gene specific primers and amplification conditions as reported earlier (Majumdar et al. 2011). The RT reaction was performed by incubating 2 lg of total RNA in 20 ll reaction volume containing 20 pmol of reverse primers, 5X reaction buffer, 10 mM dNTP mix, 200 U RevertAidTM M-MuLV Reverse Transcriptase (RT) and 20 U ribonuclease inhibitor. Prior
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to adding the RT enzyme, the reaction mixture was heated at 65 °C for 5 min and then RT was added the reaction mixture was incubated at 42 °C for 50 min for cDNA synthesis. The reaction was stopped by heating at 75 °C for 5 min. Morphology and growth of transformed root lines Root segments (*2–3 cm long; 0.1–0.15 g FW) of each Ri-transformed root line, excised from 14-day old root cultures grown on solid N/5 medium, were transferred to the 20 ml N/5 medium in 9 cm Petri-plates and cultured for 28 days in the dark. Morphological characterisations of 30 Ri-transformed root lines were done on the basis of relative thickness and lateral root density (Chaudhuri et al. 2005; Alpizar et al. 2008). After 28 days, the roots were harvested and the number of lateral branches on the primary roots of each sample was recorded, expressed as the lateral root density (number of lateral roots per centimetre). For each root line ten Petri-plates were used and each experiment was repeated three times. To determine the relative thickness, transverse section of primary root segments (*4 cm long) from three randomly selected root clones of 30 root lines were done. The growth of 30 Ri-transformed root lines cultured on solid N/5 medium was studied after 28 days. Root segment (*2–3 cm long; 0.1–0.15 g FW) of each Ri-transformed root line, excised from 14-day old root cultures grown on solid N/5 medium, were transferred in 9 cm Petri-plates containing 20 ml solid N/5 medium and cultured for 28 days under the dark condition at 24 ± 1 °C temperature. After 28 days, root tissues were harvested, FW and DW were recorded and growth index (GI) was determined. Root elongation was recorded at 2 day intervals for 20 days and expressed as linear growth rate (millimeters per day) for 2 randomly chosen LBA9402 transformed root lines (RIX-23 and RIX-33). For each root line five replicates were used and each experiment was repeated three times. Extraction and analysis of trans-resveratrol by HPLC Qualitative and quantitative analysis of trans-resveratrol was done following methods reported earlier (Rodrı´guezDelgado et al. 2002). Approximately 1 gm of dried (45 °C for 10 h) root tissue was extracted with 10 ml extraction buffer [0.1 N aqueous HCl: acetonitrile (1: 5 v/v) HPLC grade; Spectrochem] and incubated at room temperature for 1 h under shaking condition (60 rpm). The filtered extract was evaporated to dryness in vacuum and the residue was resuspended in 2 ml of HPLC grade ethanol. HPLC analysis (Agilent Technologies 1260 infinity series,
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Agilent, USA) was performed following method described earlier (Malovana´ et al. 2001; Chen et al. 2007) with minor modification on a reverse phase Agilent Eclipse Plus C-18 column (3.5 lm, 4.6 mm 9 100 mm) (Agilent, USA) with a flow rate of 0.5 ml min-1. Gradient elution was performed with solvent A consisting of methanol: acetic acid: water [10:2:88 (v/v/v)] and solvent B comprising of methanol: acetic acid: water [90:2:8 (v/v/v)] for 40 min run time. Sample injection volume was 20 ll and applied linear gradient elution was, 0 min—solvent A 85 % and solvent B 15 %; 0–5 min—solvent A 65 % and solvent B 35 %; 5–15 min—solvent A 50 % and solvent B 50 %; 15–25 min—solvent A 30 % and solvent B 70 %; 25–40 min—solvent A 100 % and solvent B 0 %. UV–Vis absorption spectra were recorded online from 190 to 600 nm during the HPLC analysis. Trans-resveratrol was detected at 303 nm using a UV detector (Agilent DAD detector) and by comparison of retention times with standard samples (retention times of trans-resveratrol 12.3 min). Quantitative estimation of trans-resveratrol in the Ri-transformed root extracts was done by comparison with a calibration graph of standard trans-resveratrol (Sigma-Aldrich). The standard curve was constructed by plotting peak areas versus amount of standard compound injected. Over a range of 2.0–0.1 lg, the relationship was linear. The samples were extracted and analyzed in triplicate with an average variation of 0.005 % in percentage trans-resveratrol recorded. Quantitative data from HPLC analysis of trans-resveratrol content in harvested root tissue expressed as milligrams per gram DW (root tissue) and productivity per Petri-plate/per flask as milligrams.
ESI–MS/MS of trans-resveratrol Purified sample of trans-resveratrol was prepared from extracted sample of hairy root tissues by preparative HPLC. Compound corresponding to trans-resveratrol peak 12.3 min (RT of standard sample of trans-resveratrol 12.3) was isolated *5 mg and was used for ESI–MS/MS. ESI–MS/MS analysis was carried out with the purified sample using a MaXis Impact (Bruker, serial number282001.00123) triple quadrupole mass spectrometer equipped with a Turbo ion spray source with the capillary voltage settled at -3600 V and heated to 200 °C. Acquisition was performed in positive ion mode in multiple reactions monitoring (MRM) using scanning range from 50 to 3000 m/z and the protonated molecule of trans-resveratrol (m/z 229) as precursor ions. The analysis with MRM was developed using as declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) the following conditions: -60, -26, -11 for transresveratrol (m/z 229).
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Time course of growth and trans-resveratrol content in Ri-transformed root lines in solid medium Time course growth study in two randomly selected root lines viz. RIX-42 and RIX-33 were determined on solid N/5 medium. Primary root segment with 4–6 laterals (*0.1 g FW) was excised from 14-day old root culture, transferred to 20 ml solid N/5 medium in 9 cm Petri-plates and cultured under the dark condition at 24 ± 1 °C temperature. There were five replicates for each root line for analysis at every 7 day interval and each experiment was repeated three times. Root tissues were harvested at 7, 14, 21, 28 and 35 day interval, dried, GI and trans-resveratrol content determined by HPLC. Establishment, growth and maintenance of Ritransformed root lines in liquid medium Excised root segments (0.1–0.15 g FW) of root line RIX33 (randomly selected among seven high trans-resveratrol yielding root lines) were inoculated in 150 ml conical flask containing 15 ml different liquid medium viz. MS medium, half strength MS medium (MS/2), and N/5 medium supplemented with or without 0.01 % activated charcoal (ACMS or MS, AC-MS/2 or MS/2 and AC-N/5 or N/5 respectively) for establishment of root cultures in liquid medium. The culture was incubated on gyratory shaker at 70 rpm for 35 days in dark at 24 ± 1 °C. For each experiment five replicates was used and each experiment was repeated three times. Root cultures of selected seven high trans-resveratrol yielding Ri-transformed root lines were established by transferring root tissue (*0.1–0.15 g FW) with 4–6 actively growing white laterals in 15 ml liquid AC-N/5 medium into the 150 ml conical flask and maintained by regular sub-culture at an interval of 4 weeks for 1 year. The culture was incubated on gyratory shaker at 70 rpm in dark at 24 ± 1 °C. NTH root culture in liquid medium was established and maintained in liquid AC-N/5 medium containing 2.5 lM IBA by regular sub-culturing at interval of 28 days. Time course of growth and trans-resveratrol content in Ri-transformed root lines in liquid medium Time course study of growth and trans-resveratrol content in liquid culture was carried out in two randomly selected high trans-resveratrol yielding root lines (root line RIX-42 and RIX-33) for 35 days. Primary roots (*2–3 cm long; 0.1–0.15 g FW) with 4–6 lateral roots were excised from the 14-day old root cultured in liquid AC-N/5 medium and inoculated in 15 ml liquid AC-N/5 medium into the 150 ml conical flask incubated on gyratory shaker at 70 rpm in
dark at 24 ± 1 °C. Root tissues were harvested at 7 days intervals to determine the FW, DW and trans-resveratrol content by HPLC. For each root line five replicates were used and each experiment was repeated three times. To compare growth and trans-resveratrol content in between root lines maintained in liquid medium, primary roots (*2–3 cm long; 0.1–0.15 g FW) with laterals were excised from stock cultures of seven selected high yielding root lines growing in liquid AC–N/5 medium for 14 days and cultured in 150 ml conical flask containing 15 ml liquid AC-N/5 medium. The cultures were incubated on gyratory shaker at 70 rpm in dark at 24 ± 1 °C. Root tissue was harvested after 28 days to determine GI and transresveratrol content by HPLC. Statistical analysis All of the experiments were randomized and were repeated at least three times. Data were examined by a one-way analysis of variance (ANOVA) to detect significant differences (p B 0.05) in the mean (Sokal and Rohlf 1987). A post hoc mean separation was performed by the Duncan multiple range test (DMRT) at the same 5 % probability level using SPSS software (version 16.0). Variability in the data was expressed as the mean ± standard deviation (SD).
Result Effect of explant type on hairy root induction in A. hypogaea cv. JL-24 Four types of explant viz. excised leaflet, petiole, internode and cotyledon, excised from 20 to 22-day old seedlings, were used to study the effect of explant type on hairy root induction following infection with A. rhizogenes strain LBA9402. Excised leaflet and petiole explants showed significantly (p B 0.05) higher frequency of root induction (88.57 ± 4.96 % and 77.19 ± 7.82 % respectively) compared to excised internode (43.75 ± 2.34 %) and cotyledon explants (15.38 ± 4.56 %). In excised leaflet explants, roots were formed mainly along the midrib region and a lesser number of root inductions were noted at the leaflet margin. The roots were whitish in color, thin or thick, highly branched and plagiotropic. Infectivity of different A. rhizogenes strains on hairy root induction The two highly susceptible explants viz. leaflet and petiole excised from 20 to 22-day old seedlings of A. hypogaea cv. JL-24, were used for the subsequent study of rhizogenic response (root induction efficiency) towards five agropine
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type strain of A. rhizogenes. No root induction was observed in any of the uninfected control explants even after 4–8 weeks of culture (Fig. 1a, b). Root induction was noted in both types of explants (Fig. 1c–f) from the pricked sites after infection with the five agropine type strains of A. rhizogenes within 10–22 days of infection (Table 1). Induction of putatively transformed roots at the responding wound sites occurred either indirectly via callus formation (Fig. 1c, d) or directly from the explants (Fig. 1e, f). In case of indirect root regeneration, appearance of friable white callus was followed by emergence of putatively transformed roots within 13–18 days. The parameters used to evaluate efficiency of hairy root induction in two types of explants of A. hypogaea cv. JL24, were time taken for hairy root initiation (days per total explants), hairy root induction percentage per total explants and hairy root induction frequency per single explant. Explants infected with strains LBA9402 and A4 showed earliest root induction i.e. within 10–13 days of infection, while explants infected with strains R1000, HRI and ATCC 15834 showed root induction after 20–23 days of infection (Table 1). The hairy root induction percentage per total leaflet/ petiole explants varied significantly (p B 0.05) depending on the strain of A. rhizogenes used for transformation. A. rhizogenes strain LBA9402 showed maximum frequency of root induction in both types of explants, while percentage of explants showing root induction was low with other strains (Table 1). The effect of A. rhizogenes strains on number of hairy root induced per explant after 28 days of infection was also studied. In both leaflet and petiole explants maximum number of root induced per explant (7.22 ± 0.67 and 8.08 ± 0.66 respectively) was obtained after infection with A. rhizogenes strain LBA9402 (Table 1).
One hundred and fifty Ri-transformed root lines of A. hypogaea were established following infection with three strains (50 root lines/strain) of A. rhizogenes, viz. strain LBA9402, strain A4, and strain R1000 of A. rhizogenes on phytohormone free MS medium containing 500 mg l-1 ampicillin within 8 weeks of infection. Ri-transformed root lines showed better growth (FW basis) on solid N/5 basal medium (GI 12.54 ± 0.87 in root line RIX-33) as compared to MS medium (GI 9.06 ± 0.45 in root line RIX-33) after 28 days. Thirty fast growing Ri-transformed root lines of A. hypogaea cv. JL-24 were selected (14 root lines transformed with strain LBA 9402, 10 root lines transformed with strain A4 and 6 root lines transformed with strain R1000) and maintained on solid N/5 medium by regular 4 weekly sub-culture for over 3 years in vitro.
Establishment, selection and maintenance of Ritransformed rhizoclones
PCR and RT-PCR analysis 30 Ri-transformed root lines revealed the presence and expression of the rolA, rolB, rolC, rolD (of TL-DNA) in all the Ri-transformed root lines irrespective of strain of A. rhizogenes used for transformation, whereas aux1 and aux2 genes (of TR-DNA) were not detected in majority of Ri-transformed root lines. Presence of amplified products of expected size in all the transformed root lines identical with the positive control, confirmed the integration, retention and expression of respective genes in the LBA9402, A4 and R1000 transformed roots (Fig. S1a– d). No amplification was observed in genomic DNA of non transformed (NTH) root culture. No amplification product of VirD1 (450 bp) was detected in any of the transformed root lines confirming that the detection of rolA, rolB, rolC, rolD, aux1 and aux2 genes in Ri-transformed root lines was not resulting from A. rhizogenes contamination.
Separate root lines were established from individual putatively transformed primary root induced at pricked site following infection with five strains of A. rhizogenes and cultured on MS medium supplemented with 500 mg l-1 ampicillin. The putatively transformed root lines established following infection with A. rhizogenes strain LBA9402, strain A4 and strain R1000 grew rapidly and developed profuse laterals within 2 weeks. Roots induced following infection with strains HRI and ATCC 15834 of A. rhizogenes, when excised and cultured on MS medium containing 500 mg l-1 ampicillin, grew slowly with few laterals and gradually turned brown. Thus, transformed root lines could not be obtained with these two strains.
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Establishment and maintenance of excised nontransformed root cultures of A. hypogaea Excised root segments derived from 20 to 22-day old axenic seedling of A. hypogaea cv. JL-24 cultured on phytohormone unsupplemented MS and N/5 medium did not grow and necrosed within 14 days. Excised root segments showed very slow growth rate and without lateral root initiation on MS medium supplemented with 0.49 or 2.5 lM IBA. When cultured on N/5 medium supplemented with 2.5 lM IBA, NT roots showed comparatively better growth rate without any callus formation. The non-transformed root cultures (NTH) of A. hypogaea were thin (\ 1 mm in diameter), slow growing (GI 1.97 ± 0.17) with lateral density of 1.25 ± 0.31 per cm even in phytohormone supplemented medium. The morphological characters were stably maintained for over 3 years on N/5 medium with 2.5 lM IBA. Confirmation of transformation
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Fig. 1 Response of explants of A. hypogaea cv. JL-24 to infection with A. rhizogenes strain LBA9402 after 14 days a control leaflet and b petiole explant; c indirect regeneration of transformed roots from
callus induced on leaflet explant and d petiole explants; e induction of transformed roots directly on leaflet explant and f on petiole explants. Bar = 0.4 cm
Morphology of transformed root lines
hormone free solid N/5 medium. However, root hairs were not observed in any Ri-transformed root lines. All of the 30 root lines could be distinguished morphologically on the basis of relative thickness and lateral root density. Relative thickness and lateral root density of the transformed root lines varied from 0.54 ± 0.07 (root line R1000-9) to
In general, all selected Ri-transformed root lines showed typical Ri-transformed phenotype. The long primary roots of each root line were with numerous laterals showing a high degree of branching and rapid, plagiotropic growth on
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Plant Cell Tiss Organ Cult Table 1 Effect of types of explant and strains of Agrobacterium rhizogenes on hairy root induction in Arachis hypogaea cv. JL-24 after 28 days of infection (n = 180 for leaflet explants and n = 90 for petiole explants) Agrobacterium rhizogenes strains (MS ? Ampicilin 500 mg l-1)
Type of explant
Time taken for hairy root initiation (days per total explants)
Hairy root induction percentage per total explants
Hairy root induction frequency per single explant
No bacteria
Excised leaflet
No root induction
0a
0a
LBA9402
10–13
A4 R1000 ATCC 15834 Excised petiole
LBA9402
7.22 ± 0.67d
88.57 ± 4.96
e
11–14
66.70 ± 10.47
3.67 ± 0.35c
20–23
b
6.27 ± 1.07d
d
2.81 ± 0.62c
c
1.61 ± 0.63b
15.72 ± 4.79
19–22
HRI No bacteria
f
44.75 ± 5.05
20–23
32.08 ± 6.72
No root induction
0p
10–13
0p
77.19 ± 7.82
s r
s
8.08 ± 0.66 r
A4
10–13
54.37 ± 15.60
4.60 ± 0.83
R1000
20–23
7.38 ± 3.46p
1.56 ± 0.51q q
ATCC 15834
20–22
38.83 ± 9.51
1.32 ± 0.20q
HRI
20–23
9.17 ± 3.19p
1.67 ± 0.27q
Values followed by same letters are not significantly different at p B 0.05 according to ANOVA and DMRT for each data point
Fig. 2 Morphological variation in 30 Ri-transformed root lines of A. hypogaea cv. JL-24
1.54 ± 0.1 mm (root line RA4-51) and 7.60 ± 0.30 (root line RIX-21) to 4.5 ± 0.5 (root line RA4-12) per cm respectively (Figs. 2, 3). Each root line and the rhizoclones derived from them stably retained the morphological character for over 3 years of maintenance in vitro. There was no correlation between presence/absence of aux1 and aux2 genes and morphology of Ri-transformed roots of A. hypogaea. Variability in growth of transformed root lines In comparison to NT roots which showed no growth and NTH roots which showed slow growth, the 30 fast growing Ri-transformed root lines showed significant increase
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(p B 0.05) in growth rate and biomass accumulation after 28 days of culture. Ri-transformed root lines showed fivefold (Root line R1000-9) to tenfold (root line RA4-19) increases over NTH root culture (GI 1.97). Fourteen LBA9402 transformed root lines showed variation in GI (FW basis) from 11.1 ± 1.16 (root line RIX-6) to 17.75 ± 1.11 (root line RIX-42) after 28 days of culture (Fig. S2a). Among the ten A4 transformed root lines, GI (FW basis) varied from 13.06 ± 0.82 (root line RA4-2) to 17.79 ± 1.35 (root line RA4-19), while GI (FW basis) of R1000 root lines varied from 9.16 ± 1.1 (root line R1000-9) to 14.18 ± 0.79 (root line R1000-8). GI (DW basis) of LBA9402 transformed root lines varied significantly (p B 0.05) from 11.73 ± 0.87 (root
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Fig. 3 a–c Ri-transformed root line RIX-2 showing a, b low lateral root density (bar = 0.5 cm), c thin diameter; d–f Ri-transformed root line R1000-8 showing d, e high lateral root density (bar = 0.5 cm) f thick diameter
line RIX-6) to 19.01 ± 1.15 (root line RIX-42). In case of A4 transformed root lines, GI varied from 19.46 ± 1.78 (root line RA4-19) to 14.05 ± 1.11 (root line RA4-2). GI of the six R1000 transformed root lines varied from 15.83 ± 1.26 (root line R1000-8) to 10.77 ± 0.95 (root line R1000-9) after 28 days of culture (Fig. S2b). The Ri-transformed root lines are characterized by vigorous root tip elongation, unlike non-transformed root cultures. Root tip elongation in fourteen LBA9402 transformed root lines varied from 6.48 ± 0.48 cm (root line RIX-25) to 8.83 ± 0.49 cm (root line RIX-41) in 28 days (Fig. S3). In ten A4 transformed root lines, root tip elongation varied from 6.56 ± 0.80 cm (root line RA4-2) to 10.43 ± 0.67 cm (root line RA4-8) while among six R1000 transformed root lines, it varied from 5.54 ± 0.53 cm (root line R1000-9) to 7.57 ± 0.47 cm (root line R1000-1) after 28 days of culture. Thus, Ritransformed root lines show significant variation in growth rate irrespective of A. rhizogenes strain used to establish the transformed root cultures in A. hypogaea. Primary root tip elongation rate (mm per day) in two randomly selected root lines RIX-23 and RIX-33 showed reproducible growth kinetics with distinct lag phase of 4–8 days, followed by log phase of 10–14 days and stationary phase after 16 days in solid N/5 medium (Fig. S4). Primary root tip elongation started within 2 days of inoculation, followed by the appearance of lateral roots within
6–8 days. Root lines RIX-23 and RIX-33 showed maximum linear growth rates 4.71 ± 0.25 mm day-1 at 12–14 days and 4.43 ± 0.33 mm day-1 at 10–16 days respectively. After 16 days of inoculation, rate of linear primary root tip elongation gradually decreased. Root lines RIX-33 and RIX-23 showed total root tip elongation of 7.75 ± 0.13 and 8.00 ± 0.25 cm, which was significantly (p B 0.05) higher than NTH (3.45 ± 0.15 cm). NT roots showed no primary root tip elongation or lateral root development and gradually necrosed. After 6 days, root tip elongation was observed in NTH roots (i.e. non-transformed root cultured on solid N/5 medium supplement with 2.5 lM IBA) and continued at very slow rate (0.93 ± 0.26 to 1.14 ± 0.35 mm day-1) from 10–20 days (Fig. S4). Identification of trans-resveratrol by ESI–MS/MS The HPLC analysis of 2 years old 30 Ri-transformed root lines, established with three strains of A. rhizogenes, revealed that trans-resveratrol was synthesized by all Ritransformed root lines, irrespective of A. rhizogenes strain used. Compound corresponding to peak 12.3 min (RT of standard sample of trans-resveratrol 12.3) was isolated *5 mg by preparative HPLC (Fig. 4) and was identified as trans-resveratrol by ESI–MS/MS. Spectrum of ESI–MS showed the characteristic peak of protonated trans-
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Fig. 4 Chromatogram of Ri-transformed root extract of A. hypogaea cv. JL-24 showing a Overlay spectrum (kmax = 303 nm) of standard transresveratrol and sample; b UV–Vis absorption spectra of isolated trans-resveratrol
resveratrol (m/z 229) as the system run in positive ionization mode. The product ion scan of the major peak at m/ z 229 when subjected to fragmentation using collision induced dissociation (CID), produce characteristic ions 107.0509, 119.059, 135.0459, 145.0666, 165.0720 and 183.0825 for trans-resveratrol molecule (Fig. 5). The fragmentation pattern is comparable to the literature values (Montsko et al. 2008; Camont et al. 2009). Enhanced trans-resveratrol content and productivity in Ri-transformed root lines The trans-resveratrol content and productivity varied significantly (p B 0.05) among the 30 selected Ri-transformed root lines of A. hypogaea cv. JL-24. Trans-resveratrol content in fourteen LBA9402 transformed root lines varied from 0.30 ± 0.01 to 0.97 ± 0.14 mg g Dw-1. Transformed root lines RIX-19 and RIX-23 showed maximum content of trans-resveratrol (0.97 ± 0.14 and 0.96 ± 0.05 mg g Dw-1 respectively), which was *15 fold enhanced as compared to the content in NTH root cultures. Maximum productivity was observed in root line RIX-19 (0.19 ± 0.02 mg per Petri-plate). Trans-resveratrol content in ten A4 transformed root lines varied from 0.28 ± 0.07 mg g Dw-1 (*4 fold of NTH) in root line RA4-6 to 0.79 ± 0.1 mg g Dw-1 (*12 fold of NTH) in root line RA4-2. Trans-resveratrol productivity in A4 transformed root lines varied from 0.08 ± 0.01 mg (root line RA4-19) to 0.16 ± 0.03 (root line RA4-2). In six R1000 transformed root lines, trans-resveratrol content varied from 0.27 ± 0.03 mg g Dw-1 (*4 fold of NTH) in root line R1000-1 to 0.73 ± 0.06 mg g Dw-1 (*11 fold of NTH) in root line R1000-2. Trans-resveratrol
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productivity in R1000 transformed root lines varied from 0.06 ± 0.01 mg (root line R1000-1) to 0.17 ± 0.01 mg (root line R1000-8). Thus, trans-resveratrol content was significantly higher (4–14 fold enhanced) in the selected fast growing thirty Ritransformed root lines as compared to NTH root cultures (Fig. 6). Establishment and maintenance of Ri-transformed root lines in liquid medium The clones of Ri-transformed root culture lines maintained for over 2 years in solid N/5 medium were used for optimization of cultural conditions for growth and transresveratrol production in liquid shake culture. Roots grew very slowly initially in full or half strength MS medium or N/5 medium and after 14 days of culture, roots turned yellowish-brown (Fig. 7a, b). The liquid basal medium also turned yellowish-brown in all cases. Addition of activated charcoal (0.01 %) improved the growth of Ri-transformed roots in liquid MS medium and N/5 medium. Roots were healthy, white, with high lateral branching and prominent plagiotropic growth (Fig. 7c, d). The liquid medium did not turn brown even after 6 weeks of culture. Root showed enhanced GI (15 fold DW basis) in N/5 medium supplemented with 0.01 % activated charcoal whereas addition of 0.01 % activated charcoal in MS medium improved growth to 8.5 fold (DW basis) after 28 days of roots culture (Fig. 8). Non-transformed root culture established and maintained in liquid N/5 medium supplemented with 2.5 lM IBA and 0.01 % charcoal, showed slow growth of roots with very few laterals (Fig. 8).
Plant Cell Tiss Organ Cult Fig. 5 ESI-MS/MS analysis of isolated trans-resveratrol showing a Mass spectrum of trans-resveratrol (m/z = 229), b MS/MS Spectrum of fragmented ion products of the peak m/z 229
Growth kinetic study in relation to trans-resveratrol content in two high yielding Ri-transformed root lines The transformed root lines RIX-42 and RIX-33 showed a lag phase of approximately 7 days, followed by log phase of growth (7–21 days) and then stationary phase (after 21 days) on both solid and liquid medium (Fig. 9). Optimum growth was obtained on day 21 in both medium after which growth rate decreased. After 21 days, ca 17–19 fold (FW basis) increase in growth was obtained in liquid medium. Biomass accumulation in root line RIX-42 was 2.16 ± 0.11 g (FW) in liquid medium which was 1.3 fold
more than in solid medium (FW 1.83 ± 0.06 g) after 35 days. Optimum accumulation of trans-resveratrol was obtained at 28 days. Thus, the trans-resveratrol production in Ri-transformed root was growth dependent and maximum at the end of growth phase. Production of trans-resveratrol in selected Ritransformed root lines in liquid medium Growth, biomass accumulation, trans-resveratrol content and productivity was studied in seven high yielding (transresveratrol content [0.6–1.0 %) Ri-transformed root lines viz. root line RIX-18, RIX-19, RIX-23, RIX-33, RA4-2,
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Fig. 6 Quantitative analysis of trans-resveratrol of 30 Ri-transformed root lines cultured on solid N/5 medium for 28 days a transresveratrol content (in mg g Dw-1), b productivity of trans-
resveratrol per Petri-plate (in mg). Bars with the same letters are not significantly different at p B 0.05 according to ANOVA and DMRT for each data point
RA4-8, and R1000-2 in liquid AC-N/5 medium after 28 days (Fig. 10a–c). The GI (DW basis) varied significantly (p B 0.05) among seven Ri-transformed root lines from 18.91 ± 0.82 to 24.98 ± 1.02. Optimum biomass accumulation was obtained in root lines R1000-2 (FW 2.1 ± 0.13 g per flask and DW 0.375 ± 0.015 g per flask). Among the seven selected Ri-transformed root lines, optimum trans-resveratrol content (1.21 ± 0.09 mg g Dw-1) and productivity (0.37 ± 0.08 mg per flask) which was 19 fold higher than NTH root culture (Fig. 10b, c).
Discussion
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Hairy root culture is an effective, low cost and simple method in modern biotechnology. For last few decades, transformed hairy roots have become a valuable tool for the production of secondary metabolites and engineering of metabolic pathways. Hairy root cultures have been established from important medicinal plants. In the present study, we describe an efficient protocol of A. rhizogenes mediated transformation of peanut cv. JL-24, a popular
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Fig. 7 Growth of Ri-transformed root line RIX-33 in liquid medium after 28 days a in MS medium, b in N/5 medium, c in MS medium supplemented with 0.01 % activated charcoal, d in N/5 medium supplemented with 0.01 % activated charcoal. Bar = 0.5 cm
Indian cultivar for production of trans-resveratrol. Present investigation showed that type of explant and A. rhizogenes strain used greatly influenced hairy root induction in A. hypogaea cv. JL-24. In the present study, three parameters (hairy root initiation days per total explants, hairy root induction percentage per total explants and hairy root induction frequency per explant) were used to estimate the hairy root induction efficiencies of different strains of A. rhizogenes on different type of explants. The leaflet and petiole explants of A. hypogaea cv. JL-24 showed significantly higher root induction frequency (88.57 ± 4.96 and 77.19 ± 7.82 % respectively) than other type of explants with strain LBA9402. Of the five A. rhizogenes strains used, strain LBA9402 showed maximum infectivity as compared to strains A4, R1000, ATCC 15834 and HRI. Root induction potential of the various strains of A. rhizogenes vary significantly due to differential virulence of various strains and genotype specificity (Chandran and Potty 2008; Setamam et al. 2014).
The Ri-transformed root lines of A. hypogaea cv. JL-24 established and maintained in vitro in the present study, showed high degree of branching and rapid, plagiotropic growth on phytohormone free solid N/5 medium but were devoid of root hairs as reported earlier in peanut (MedinaBolivar et al. 2007) and Tylophora indica (Chaudhuri et al. 2005). Lateral root density in Ri-transformed root lines of A. hypogaea cv. JL-24 was significantly higher (7.6 ± 0.3 to 4.5 ± 0.5) as compared to NTH root culture (1.25 ± 0.31). The precise mechanisms involved in lateral root formation are still not clearly understood, but roots transformed by A. rhizogenes are characterized by the spontaneous formation of numerous laterals, an important factor contributing to their high biomass productivity. Most of the large-seeded legumes in general and peanut in particular, are recalcitrant to regeneration and genetic transformation (Geng et al. 2012), as such A. rhizogenesmediated transformation provides a system for development of composite plants with transgenic roots. Several researchers successfully produced composite/chimeric
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Fig. 8 Comparative growth study (on the basis of FW) of two Ritransformed root lines RIX-33 and RIX-42 on solid N/5 medium, liquid N/5 medium and liquid AC-N/5 medium after 28 days. Bars with the same letters are not significantly different at p B 0.05 according to ANOVA and DMRT for each data point
peanut plant after infection with A. rhizogenes with different objectives that includes the investigation of root nodule formation (Sinharoy et al. 2009; Akasaka et al.
1998) and disease resistance (Geng et al. 2012). None of them studied the resveratrol content in roots of composite plant which assigns ample scope for further studies. Medina-Bolivar et al. (2007) and Condori et al. (2010) reported induction and establishment of transformed root cultures with A. rhizogenes strain ATCC 15834 in two different varieties of A. hypogaea using cotyledonary node/ leaf as explant. There are few studies on trans-resveratrol content in Ri-transformed root lines in A. hypogaea. Medina-Bolivar et al. (2007) reported that intracellular trans-resveratrol content of two untreated ATCC 15,834 transformed root clones of A. hypogaea cv. Andru II varied from 0.21 to 0.58 mg g Dw-1 in liquid culture. Condori et al. (2010) studied the effects of culture medium and growth stage on induced biosynthesis of trans-resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3. Sodium acetate (elicitor) treatment and incubation of 24 h showed secretion of stilbenoids into the media by hairy roots. In our present study, we found that 18-month old Ritransformed root lines, that were established following the infection of three strains of A. rhizogenes, showed significant (p B 0.05) variation in trans-resveratrol content from 0.27 ± 0.03 to 0.97 ± 0.14 mg g Dw-1 after 28 days of culture in solid N/5 medium, which was significantly higher than the content in non-transformed root cultures growing in phytohormone supplemented basal medium. This is the first report of transformation of A. hypogaea
Fig. 9 Time course study of fresh weight (FW) biomass and trans-resveratrol content in solid and liquid cultures of two selected root lines RIX33 and RIX-42 over a period of 35 days
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Fig. 10 a Growth study (FW and DW basis) of seven selected Ritransformed root lines cultured in liquid AC-N/5 medium after 35 days; b, c quantitative analysis of trans-resveratrol of seven selected Ri-transformed root lines cultured in liquid AC-N/5 medium after 35 days b Trans-resveratrol content (mg g Dw-1); c productivity of trans-resveratrol (in mg) per flask. Bars with the same letters are not significantly different at p B 0.05 according to ANOVA and DMRT for each data point
with A. rhizogenes strain LBA9402 and production of high levels of trans-resveratrol in LBA9402 transformed root lines. Our result showed a significant variation in morphology, growth and trans-resveratrol content in between the 30 selected root lines studied; this variation in growth, secondary metabolite content in between transformed root lines have been reported earlier in Atropa belladonna, Catharanthus roseus, Przewalskia tangutica, Rauvolfia serpentina, Tylophora indica (Aoki et al. 1997; Batra et al. 2004; Chaudhuri et al. 2005; Lan and Quan 2010; Ray et al. 2014; Roychowdhury et al. 2015). Since each root line is
resultant of a single transformation event and derived from a single transformed cell, the variability among different root lines has been attributed to the nature of the T-DNA integration into the host genome, physiological state of transformed cell, copy number of T-DNA inserted and differential expression of rol genes etc., (Jouanin et al. 1987; Moyano et al. 1999; Chandra 2012; Roychowdhury et al. 2015). Hence in the present study insertion of Ri T-DNA in transformed root lines of A. hypogaea cv. JL-24 ultimately result a stimulatory effect on trans-resveratrol content irrespective of A. rhizogenes strain used. Variability in biomass accumulation and trans-resveratrol content in transformed root lines of A. hypogaea indicates that characterisation and selection of fast growing, high yielding transformed root line/lines were crucial for secondary metabolite productivity in transformed hairy root cultures of any species as has been demonstrated in cultured plant cells of different species (Deus and Zenk 1982; Holden et al. 1988). In general, hairy roots can grow on any of the basal medium used in tissue culture, yet the nutritional requirements vary with the species/varieties studied and media composition can have a significant impact on root growth in culture systems (Sivakumar et al. 2005). In A. hypogaea, composition of medium seems to play an important role depending on the cultivar used. Medina-Bolivar et al. (2007) used Gamborg’s B5 medium to grow peanut hairy roots of cv. Andru II while Condori et al. (2010) later reported that a modified MS (MSV medium that differs from MS and B5 media in the ratio of ammonia to nitrate concentration) medium resulted in higher root biomass when compared to B5 medium. In our present study, optimum and sustainable growth of hairy root culture of A. hypogaea cv. JL-24 was achieved in N/5 medium (modified MS medium containing 1/5th of total nitrogen of the normal strength, macro nutrients, micro nutrients, myoinositol, ammonium iron citrate, filter sterilized Gamborg’s vitamin) in solid culture. While in liquid culture, N/5 medium alone was not suitable for root growth and was supplemented with 0.01 % activated charcoal. Activated charcoal has often used in plant tissue culture, in both liquid and semi-solid media, to improve the growth and development by the adsorption of inhibitory substances in the culture medium (Weatherhead et al. 1979; Pan and Staden 1998), drastic decrease in the phenolic oxidation or brown exudate accumulation (Madhusudhanan and Rahiman 2000). By optimizing hairy root culture growth and culturing conditions, biomass accumulation in A. hypogaea transformed root line RIX-42 was enhanced 1.3 fold in liquid medium as compared to solid medium. Seven transformed root lines which were high trans-resveratrol yielding ([0.6 mg g Dw-1) could be selected and cultured in liquid medium. All of the root lines
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showed optimum intra-cellular trans-resveratrol content at stationary phase of growth. Among the seven Ri-transformed root lines, root line RIX-19 showed maximum accumulation and productivity of trans-resveratrol (1.21 ± 0.09 mg g Dw-1 and 0.37 ± 0.08 mg per flask respectively), which was 19 fold higher than non-transformed (NTH) root cultures. The production of stilbenes from plants, in general, is often restricted to a given species or genus and might be activated only during a particular development stage or underspecific seasonal, stress, or nutrient availability conditions (Verpoorte et al. 2002). Due to stereospecificity and the high costs involved in chemical synthesis of these compounds much effort has been put into the use of in vitro cultures as one attractive biotechnological strategy for producing this natural compound of commercial interest (Almagro et al. 2013; Jeandet et al. 2014). Due to genetic and biosynthetic stability, cost efficiency and amenability for scale up cultivation in bioreactors (Ono and Tian 2011; Roychowdhury et al. 2013), hairy root has now been recognized as the most proficient alternative for serving as sources of valuable phytomolecules of industrial demand (Gupta et al. 2015). Sivakumar et al. (2010) reported biomass production of hairy roots of A. hypogaea cv. Hull and Artemisia annua in scaled up mist bioreactor of 20 L. Different inexpensive elicitors and different bioreactors can be used further for industrial production of phytochemicals by increasing yields and driving down production costs. Recently Yang et al. (2015) showed that cotreatment with methyl jasmonate and cyclodextrin enhanced production of resveratrol, piceatannol, arachidin1 and arachidin-3 in hairy root cultures of peanut cv. Hull established previously by the Medina-Bolivar laboratory. In the present study, transformation of A. hypogaea with A. rhizogenes for selection of high trans-resveratrol yielding ([0.6 mg g Dw-1) root lines and optimization of culture condition in liquid shake culture for enhanced (19 fold) trans-resveratrol production in root line RIX-19 suggests that the high trans-resveratrol producing Ri-transformed root lines can be used for further scale-up studies in bioreactors. Besides this, peanut hairy root system provides a promising platform for identification of genes involved in stilbenoid biosynthesis and for study of regulation of the stilbenoid biosynthetic pathway. Acknowledgments MH is grateful to the University Grants Commission, New Delhi, for the award of Junior (Sanction No.—UGC/ 1015/Jr. Fellow (Sc.); date-15.10.09) and Senior Research Fellowship (sanction No.—UGC/430/Jr. Fellow; date-16.04.12) and expresses his sincere gratitude to Prof M. Dasgupta, Department of Biochemistry, C.U., for her guidance and encouragement. Thanks are due to Mr. Sudip Saha for his help in HPLC analysis. We also thank the Coordinator, DBT-IPLS, University of Calcutta for providing ESI–MS/MS facilities.
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Author contribution MH and SJ conceived and designed research.MH did all the experiments and analyzed the data. SJ and MH wrote the final manuscript. All authors read and approved the manuscript. Compliance with ethical standards Conflict of interest of interest.
The authors declare that they have no conflict
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