Improving hairy root induction of Urena lobata L by Agrobacterium rhizogenes ATCC 15834 by some factors

Science & Technology Development Journal, 21(3):90- 97 Original Research University of Science, VNU-HCM, Ho Chi Minh City, Viet Nam Correspondence Quach Ngo Diem Phuong, University of Science, VNU-HCM, Ho Chi Minh City, Viet Nam Email: qndphuong@hcmus.edu.vn History  Received: 11 July 2018  Accepted: 05 November 2018  Published: 13 November 2018 DOI : https://doi.org/10.32508/stdj.v21i3.430 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Improving hairy root induction of Urena lobata L. by Agrobacterium rhizogenes ATCC 15834 by some factors Vu Thi Bach Phuong, Pham Thi Anh Hong, Quach Ngo Diem Phuong ABSTRACT Introduction: Our previous study showed that Urena lobata L. hairy root is a potential pharma- ceutical source for type 2 diabetes treatment. In order to improve the transformation efficacy and the quality of hairy roots, this study examined the effects of several factors including age, parts of plants, infection time and culture medium in inducing hairy roots in Urena lobata L.Methods: In this study, we investigated four factors to improve the hairy root induction in Urena lobata L. These factors include: age of plant (15-day-old in vitro plants, 45-day-old in vitro plants and after two sub- culture generations plants), different parts of plant (roots, stems, and leaves), infection time (10, 20 and 30 minutes), and culture medium (Murashige and Skoog (MS), Gamborg B5 medium (GB5) andWoody plantmedium (WPM)). All experiments were repeated three times, with uninfected leaf explants of 15-day-old in vitro as the negative control. The transformation frequency and the fresh biomass of hairy roots were recorded at four weeks after infection. Results: The results showed that the optimized procedure which used 15-day-old in vitro plants, the leafy part, the infection time of 10 minutes and culture in the WPM medium was better than the original procedure. The optimized procedure achieved a transformation frequency of 100%. In addition, the fresh biomass of hairy roots formed on an explant in the optimized procedure was 3.2 times higher than the ones induced by the original procedure. Conclusion: The results showed that the optimized procedure was more effective than the original procedure in inducing Urena lobata hairy roots. Key words: Agrobacterium rhizogenes, Hairy root, Induction, Infection, rolB, rolC, virG, Urena lobata L. INTRODUCTION Urena lobata L. belongs to the Malvaceae family, which is used in herbal medicine to treat a wide range of ailments such as colic, malaria, gonorrhea, fever, wounds, toothache, rheumatism 1, and especially diabetes 2. Studies have shown that the extracts from Urena lobata L. have medicinal activities such as antioxidant, anti-inflammatory, antimicrobial, antidiarrheal, antidiabetic, anti-hyperlipidemic, and anti-diarrhoeal activities 1,3. In addition, studies on phytochemical have analyzed and identified different compounds from Urena lobata L. extracts such as alkaloids, falconoids, tannin, saponin, coumarin, steroid/triterperioid, furocoumarin, mangiferin, quercetin, imperatorin, -sitosterol, kaempferol, luteolin, hypolatin, gossypetin, and stigmasterol 4–6. In plant tissue culture, hairy root culture technique is a key step in the production of secondary com- pounds in vitro. Hairy roots are generated by in- fecting Agrobacterium rhizogenes T-DNA into the genome of the plant. The conditions of the gene trans- fer (the nature and age of the plants, the bacterial strain, the bacterial density, and the infection pro- cess) have a great influence on the frequency of gene transfer as well as the growth and yield of the hairy roots. For examples, hairy root induction in Agas- tache foeniculum, Rosmarinic acid content in trans- formed roots (213.42 g/g dry wt) was significantly higher than non-transformed roots (52.28 g/ g dry wt) 7. Moreover, hairy root induction in Hypericum perforatum L., ATCC15834 strain and the excised seedling as explant produced the highest number of hairy roots 8. After optimizing culturing parameters (medium composition, elicitor, precursor), hairy roots can be optimized to grow rapidly and produce valu- able compounds 9. Due to the medicinal properties of Urena lobata L. and the advantage of hairy root culture technique, this study aimed to optimize hairy root induction in in vitro culture of Urena lobata L. to produce highly bioactive materials for the pharmaceutical industry. Therefore, this study focused on examining factors (age, plant parts, infection time, and culture medium) affecting hairy root induction to improve the hairy root induction and to increase the transformation fre- quency of Urena lobata L. Cite this article : Phuong V T B, Hong P T A, Phuong Q N D. Improving hairy root induction of Urena lobata L. by Agrobacterium rhizogenes ATCC 15834 by some factors. Sci. Tech. Dev. J.; 21(3):90-97. 90 Science & Technology Development Journal, 21(3):90-97 METHODS Chemicals Taq polymerase, 100 bp Plus Ladder were purchased from Bioline. rolB, rolC, virG were produced by Inte- grated DNA Technologies. Sterilization of in vitro culture materials Seeds of Urena lobata L.was locally collected in dis- trict 9, Ho Chi Minh City, Vietnam. The selected seeds had good quality and free from infection. The seeds were washed with 80% ethanol for twominutes. Then, the seedswere shaken and soaked in 2% sodium hypochlorite for 10 minutes and then washed with sterile distilled water. Seeds after sterilization were placed on the MS (Murashige and Skoog) medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) phytoagar (pH 5.8). The seeds germinated in a growth chamber at 25 2oC under standard cool white fluo- rescent tubes with a 16-h/8-h photoperiod. Plantlets were collected at different ages depending on the pur- pose of the experiment. Investigating factorsaffectinghairy root in- duction in Urena lobata L. Preparation of Agrobacterium rhizogenes Agrobacterium rhizogenes ATCC15834 strain was ob- tained from RIKEN bank (Japan) through the MEXT project. A. rhizogenes ATCC15834 cells were grown in a nutrient broth medium (beef extract 3 g/L, pep- tone 5 g/L, pH 7.0) for 48 hours in a shaking incubator (110 rpm, 25 1oC). The original procedure for hairy root induc- tion The original procedure used for Urena lobata L. hairy root induction was as following 10: the in vitro leaves from 15-day-old plants were injured on the surface to facilitate the infection process. These segments were soaked in the A. rhizogenes ATCC15834 suspension (OD600 = 0.6) for 20 minutes. After four days of co- cultivation, the explants were transferred to the MS medium (3% sucrose) supplemented with cefotaxime (250 mg/L) to eliminate the remained A. rhizogenes ATCC15834. The samples were grown under dark conditions at 25oC for hairy root induction. Within two weeks, numerous hairy roots emerged from the wounded sites on leaf explants. The number of re- sponsive explants and number of hairy roots per ex- plant were recorded 30 days after infection. Besides the investigated factors, the remaining factors were similar to the original hairy root induction pro- cedure. All experiments were repeated three times, with the same negative control is leaf explants from 15-day-old in vitro seedlings that were not infected with Agrobacterium rhizogenes. The effect of age and different parts of plantson hairy root induction Roots, stems, leaves of three types of plants: 15-day- old in vitro plants, 45-day-old in vitro plants and after two subculture generations plants were infected with Agrobacterium rhizogenes to induce hairy roots. The effect of infection time on hairy root in- duction Samples were infected with Agrobacterium rhizogenes for 10 minutes, 20 minutes, and 30 minutes to deter- mine the optimal infection time. Theeffectof culturemediumonhairy root in- duction Three types of medium were used: MS, GB5 (Gam- borg B5medium) andWPM to determine the optimal culture medium. The effect of the combined of improved fac- tors on hairy root induction The hairy roots were induced in two procedures: the original procedure and the optimized procedure with optimized conditions (age, plant parts, time of infec- tion and medium of induction). Confirmation of transgenic roots GenomicDNA sampleswere extracted fromUrena lo- bata L. hairy roots and in vitro roots (non-transgenic roots) by the CTAB method as described previ- ously 11. The Ri-plasmid was isolated from A. rhi- zogenes ATCC 15834 by the method described by Curier and Nester 12. PCR reactions were per- formed using the genomic DNA from the hairy roots and non-transgenic roots as well as the Ri- plasmid with specific primer sets for rolB, rolC, and virG genes. The sequencing primers in- clude F-rolB (5’- GCTCTTGCAGTGCTAGATTT- 3’), R-rolB (5’-GAAGGTGCAAGCTACCTCTC-3’); F-rolC (50-CTCCTGACATCAAACTCGTC-3’), R- rolC (5’-TGCTTCGAGTTATGGGTACA-3’); and F- virG (5’-TTATCTGAGTGAAGTCGTCTCAGG-3’), R-virG (5’-CGTCGCCTGAGATTAAGTGTC-3’). The expected amplified fragment sizes were 423 bp for rol gene, 626 bp for rolC gene, 1030 bp for virG gene 13. The PCR reactions were performed in a to- tal volume of 25l containing 100ng of plant genomic 91 Science & Technology Development Journal, 21(3):90-97 DNA (or 40 ng of Ri-plasmid DNA), 5 μL Taq poly- merase buffer (5X), 0.5 μM of each primer and 1U Taq polymerase (Bioline). PCR steps included ini- tial denaturation at 95oC for 5 minutes, followed by 35 cycles of amplification (95oC for 30s, 54oC for 30s and 72oC for 60s) and a final extension at 72oC for 10 minutes. PCR products were visualized by agarose gel electrophoresis. Statistical analysis Each treatment included 20 explants in replicates of three. The transformation frequency was calculated at four weeks after infection. All data analyses were performed using the SPSS 16.0 (Copyright SPSS Inc.). Experimental results were shown asmean standard deviation (SD). Differences betweenmeans were eval- uated by Duncan’s multiple range tests. Statistical sig- nificance was accepted at 0.05. RESULTS Sterilization of in vitro culture materials The seeds of Urena lobata L. were sterilized by 2% sodium hypochlorite in 10 minutes. The germination rate was 90-100%. After two days on MS medium, germinated seeds developed into seedlings. 45-day- old plants were mature enough and suitable for sub- culture. Seedlings were collected at different ages de- pending on the specific purposes of each experiment (Figure 1). Investigating important factors to improve the hairy root induction in Urena lobata L. The effect of ageanddifferent parts of plants on hairy root induction After four weeks after infection,Urena lobata L. leaves had the highest transformation frequency compared to the stems and roots at all ages (Table 1). The older plants had a lower transformation frequency than the younger plants. Specifically, leaves of 15-day-old in vitro plants had the highest transformation frequency (97.33%), and the second highest was stems (86.33%) of the same age plants (Figure 2). This result demon- strated that the younger tissues, especially cotyledon, positively correlate with the higher transformation frequency. The effect of infection time on hairy root in- duction The results indicated that the infection time of 10 and 20 minutes had a similar transformation frequency (97.67% and 97.33%, respectively) (Table 2). How- ever, when the infection time increased to 30 min- utes, the transformation frequency significantly de- creased (93.67%) (Table 2). In addition, the longer infection time correlates with a lower recovery rate of the wounded explants, and the induced hairy roots also developed to the lesser extent because of the bac- terial overgrowth during the co-cultivation. Explants infected for 10minutes havemore hairy roots than the ones infected for 20 and 30 minutes (Figure 3). Effect of culturemediumonhairy root induc- tion The results showed that WPM andMSmedium had a similar transformation frequency four weeks after in- fection (100% and 97.3%, respectively). GB5medium had the lowest transformation frequency (91.67%) four weeks after infection (Table 3). However, WPM medium had higher hairy root development than the other two media (Figure 4). This observation indi- cated that the composition and themineral content in WPMmedium are better in supporting the growth of the Urena lobata L. hairy roots than the MS and GB5 medium. The effect of the optimized conditions on hairy root induction After identifying the optimal conditions for the hairy root induction in Urena lobata L., these conditions were combined and compared to the original proce- dure. Specifically, 15-day-old in vitro leaves were in- fected for 10 minutes and cultured in WPMmedium. The results in the table 4 and the figure 5 showed that the optimized conditions had the transforma- tion frequency of 100%, while the original proce- dure achieved a transformation frequency of 97.33% (Table 4, Figure 5). Importantly, the fresh biomass of hairy roots per explant in the optimized proce- dure (0.517g) was 3.2 times higher than the one of the original procedure (0.160g). In summary, this result showed that the optimized procedure ismore effective than the original procedure in inducing and support- ing the growth of hairy roots in Urena lobata L.. Transgenic roots were confirmed by PCR The hairy root samples were analyzed by PCR to test whether the transgenic process was successful. DNA samples from the putative hairy roots and from in vitronon-transgenic roots were isolated and subjected to PCR analysis for the presence of rolB, rolC, and virG genes, which are present in Ri-plasmid. The Ri-plasmid of A. rhizogenes ATCC15834 was also in- cluded to serve as a positive control. The presence of 92 Science & Technology Development Journal, 21(3):90-97 Figure1: InvitroUrena lobataL. seedlings. (A) 5-day-old invitroplant, (B) 15-day-old invitroplant, (C) 45-day-old in vitro plant, (D) after two subculture generations plants. Table 1: The effects of age and parts of plants on transformation frequency four weeks after infection (%) Parts of Urena lobata L. Transformation frequency (%) Root 7.333f  2.081 Stem 6.333f  1.528 Leave 23.000e  2.645 Root 8.333f  2.081 Stem 32.667d  3.055 Leave 40.000c  3.000 Root 30.333d  2.517 Stem 86.333b  2.517 Leave 97.333a  2.082 Datawere shown asmean SDof three independent experiments. Letters a, b, c, d, e and f indicate significant differences at p < 0.05 according to Duncan’s multiplerange-posthoc tests. Table 2: The effect of infection time on hairy root induction after four weeks of infection Infection time (minute) Transformation frequency (%) 10 97.667a  2.517 20 97.333a  2.082 30 93.667b  3.215 Data were shown as mean SD of three independent experiments. Let- ters a and b indicate significant differences at p < 0.05 according to Dun- can’s multiplerange-posthoc tests. Table 3: The effect of culture medium on hairy root induction four weeks after infection Culture media Transformation frequency (%) MS 97.333a  2.082 WPM 100.000a  0.000 GB5 91.667b  1.528 Data were shown as mean SD of three independent experi- ments. Letters a and b indicate significant differences at p < 0.05 according to Duncan’s multiplerange-posthoc tests. 93 After two subculture generations plants 45-day-old in vitro plant 15-day-old in vitro plant Science & Technology Development Journal, 21(3):90-97 Figure 2: The effect of age and parts of plants on hairy root induction four weeks after infection. (A) unin- fected leaf explants (negative control). (B, C, D) explants form roots, stems, leaves of after two subculture gener- ations, (E, F, G) explants form roots, stems, leaves of 45-day-old in vitro plant, (H, I, J) explants form roots, stems, leaves of 15-day-old in vitro plant. Figure 3: The effect of infection time on hairy root induction four weeks after infection. (A) uninfected leaf explants (negative control), (B, C,D) infectedwithA. rhizogenesATCC15834 for 10, 20, and 30minutes, respectively. Table 4: The combined effect of the optimized condition on hairy root induction four weeks after infection Induced process Transformation frequency (%) Fresh weigh/explant (g) Original condition 97.333 2.082 0.160 0.053 Improved condition 100.000 0.00 0.517 0.076 94 Science & Technology Development Journal, 21(3):90-97 Figure 4: The effect of culture mediums on hairy root induction after three weeks after infection. (A) unin- fected leaf explants (negative control), (B, C, D) explants infected with A. rhizogenes ATCC15834 in MS, WPM, GB5 medium, respectively. Figure 5: The combined effect of improved factors on hairy root induction after fourweeks of infection. (A) uninfected leaf explants (negative control). (B) Hairy roots are induced by the original process. (C) Hairy roots are induced by the optimized process. rolB and rolC, as well as the absence of virG from these hairy roots confirmed that Ri-plasmid was integrated successfully into the plant genome (Figure 6). DISCUSSION The age and parts of plants are important factors in- fluencing the transformation of Agrobacterium rhizo- genes into plants. Young plants and seedlings have higher transformation efficacy than tissues and or- gans from mature plants. Young plant tissues such as hypocotyl, cotyledon, and young leaves are often used for infection to induce hairy roots 14. Our data in Urena lobata L. also showed that leaves from 15- day-old plants have the highest rate of hairy root in- duction. In 15-day-old Urena lobata L., the leaves are cotyledons, and the stem is hypocotyl. Hence, they are good materials for infection. In addition, many studies also showed that leaves have the highest rate of hairy root induction. This observation was attributed to the ability of leaves to produce a large number of cells in wound healing response. Wound healing re- sponse is the most important factor in inducing hairy roots. The optimal infection time to induce hairy root for each plant species is different. For example, the opti- mal infection time to induce hairy root in Urena lo- bata L. is 10 minutes. Likewise, the optimal infection time for Berberis aristataDC is 3 hours 15 and Arachis hypogaea L. is 20 minutes 16. Therefore, it is necessary to optimize the infection time for each plant species to achieve the highest hairy root induction. The culture medium is one of the most important fac- tors influencing the induction as well as the growth of hairy roots. In this experiment, WPM medium was the optimal media to support the induction and growth of hairy roots in Urena lobata L., which in- dicates that the composition and the mineral content in WPM medium were suitable for the growth of the Urena lobata L. hairy roots. WPM medium is fre- quently used to support the growth of woody species, whichmay support the growth of a subshrubplant like Urena lobata L. better than MS and GB5 medium. CONCLUSIONS This study identified important factors that can im- prove the hairy root induction in Urena lobata L. by 95 Science & Technology Development Journal, 21(3):90-97 Figure 6: Confirmation of transformation by PCR. PCR amplification of rolB (lanes 1, 2, 3), rolC (lanes 5, 6, 7), and virG (lanes 8, 9, 10) genes fromUrena lobata L. roots and hairy roots, andAgrobacterium rhizogenes ATCC15834. Lane M: molecular weight marker (100 bp plus ladder); lanes 1, 5, 9: Agrobacterium rhizogenes ATCC15834DNA (positive control); lanes2, 6, 8: DNA from in vitronon-transformed roots (negative control); lanes 3, 7, 10: DNAof transformed roots obtained afterAgrobacteriumrhizogenesATCC15834 infection; lane4: negative control of PCR amplification. Agrobacterium rhizogenes (ATCC 15834). These fac- tors include parts of plants, age of plants, infection time and culture medium. By using the optimized conditions (leaves of 15-day-old plants, infection time of 10 minutes, WPMmedium), 100% transformation efficacy was achieved, and the fresh weight of hairy roots per explant was 3.2 times higher than that of the original procedure. COMPETING INTERESTS The authors declare that they have no conflict of in- terest. AUTHORS’ CONTRIBUTIONS Vu Thi Bach Phuong implemented the experiment and wrote the manuscript. Quach Ngo Diem Phuong proposed ideas and reviewed. PhamThi Anh Hong is the advisor. 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