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Artemisia Tilesii Ledeb Hairy Roots Establishment Using Agrobacterium Rhizogenes-Mediated Transformation a
a
a
a
N. A. Matvieieva , A. M. Shakhovsky , V. B. Belokurova & K. O. Drobot a
Institute of Cell Biology and genetic engineering, National Academy of Sciences of Ukraine, Kyiv, Ukraine Accepted author version posted online: 02 Apr 2015.
Click for updates To cite this article: N. A. Matvieieva, A. M. Shakhovsky, V. B. Belokurova & K. O. Drobot (2015): Artemisia Tilesii Ledeb Hairy Roots Establishment Using Agrobacterium Rhizogenes-Mediated Transformation, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2015.1031393 To link to this article: http://dx.doi.org/10.1080/10826068.2015.1031393
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Artemisia tilesii Ledeb Hairy Roots Establishment Using Agrobacterium rhizogenesMediated Transformation N. A. Matvieieva1, A. M. Shakhovsky1, V. B. Belokurova1, K. O. Drobot1 1
Institute of Cell Biology and genetic engineering, National Academy of Sciences of Ukraine, Kyiv, Ukraine Corresponding author: Matvieieva Nadiia E-mail: [email protected]
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
Abstract An efficient and rapid protocol for the establishment of Artemisia tilesii “hairy” root culture is reported. Leaf explants of aseptically growing plants were co-cultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5-6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator EmistimTM on the stimulation of A.tilesii “hairy” root growth was studied. Maximum stimulating effect both for the control and transgenic roots was observed in case of root cultivation on the media supplemented with IBA - up to 7.95- and 9.1-fold biomass increase respectively. Cultivation on the medium with 10 μl/l EmistimeTM has also led to the control roots growth stimulation (up to 2.75-fold). EmistimeTM at 5 μl/l concentration led to 5.46-fold mass increase in only one “hairy” root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.
1
KEYWORDS: Artemisia tilesii Ledeb; Agrobacterium rhizogenes; “hairy” root culture; growth stimulation
INTRODUCTION Plant genetic transformation with Agrobacterium rhizogenes soil phytopathogenic
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
bacteria makes it possible to co-transform plant cells with more than one T-DNA at the same time. Agrobacterium cells can carry not only the resident A. rhizogenes Ri -plasmid containing the root locus rol genes responsible for root proliferation but also plasmids of interest carrying selective (bar, nptII and other genes) and the wide range of target genes (1-3). Using of medicinal plants for Agrobacterium rhizogenes-mediated transformation to produce compounds with medical properties in “hairy” roots is now of great interest. “Hairy” roots can be used for production of biologically active substances including secondary metabolites (4-6). They grow without addition of growth regulators and produce the metabolites of the initial plants. “Hairy” root culture are characterized also by rapid biomass increase.
Some species of Artemisia genus including A.absinthium, A.annua, A.vulgaris, A. vestita and A.dubia was studied as a natural source of secondary metabolites (7-11). Using of biotechnology approaches for antimalarial sesquiterpene artemisinin production is caused by the limited native plant resources of this genus and difficulty of its total chemical synthesis. Artemisia transgenic root culture can be an alternative for artemisinin production. Agrobacterium rhizogenes-mediated transformation method was used for A. annua (12, 13), A. dubia, A. indica (14), A. vulgaris (9) ”hairy” roots induction. At the
2
same time up to now any investigation was not carried out to establish A. tilesii “hairy” root culture. A. tilesii known as Aleutian mugwort has the high ability for adaptation to abiotic stress conditions. These plants also are characterized by antirheumatic, disinfectant, deodorant, anti-tumor effect and used in Alaska traditional medicine (15).
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
Induction and studying of A. tilesii “hairy” root culture growth was the aim of our work.
EXPERIMENTAL Plant Material And Bacteria Strains Used For Transformation Artemisia tilesii Ledeb seeds were used to induce in vitro culture. The seeds were obtained from the Polar-Alpine Botanical Garden (Kirovsk, Hibines, Russia) in 1997 and stored in the Seed Bank of the Institute of Cell Biology and Genetic Engineering (16). Artemisia tilesii plants cultured in vitro (Fig.1a) via micropropagation on hormone-free MS basal medium (17) were used for genetic transformation experiments.
Agrobacterium rhizogenes A4 wild strain and the bacteria carrying the binary vectors pCB124 (18) and pCB161 (19) were grown in LB liquid culture medium containing 10 g/l casein hydrolyzed, 5 g/l yeast extract, 10 g/l NaCl, 100 mg/l carbenicillin, 50 mg/l rifampicin, pH 7.0 overnight at 28°C in the dark on shaker (220 rpm). A.rhizogenes A4 strain was cultivated in LB liquid culture medium without antibiotics.
“Hairy” Root Induction Slightly sliced upper and lower leaf explants isolated from 30-days-old in vitro grown plants were co-cultured with A. rhizogenes suspension during 30 min, transferred to the
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agar-solidified 1/2MS medium (MS medium with twice reduced macrosalts content) in Petri dishes and cultured for 48 hrs in the dark at 28º C. Then they were transferred to the agar-solidified 1/2MS medium (20 g/L sucrose, pH 5.7) supplemented with 600 mg/L cefotaxime for agrobacteria elimination. Thirty explants were used in each experiment (the experiments were performed in triplicate). Root induction frequency was measured
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as the ratio of the number of explants with induced roots to the total number of explants used for transformation.
Initiated “hairy” roots were transferred on 1/2 MS medium supplemented with 2 % sucrose, 0.7% agar, 600 mg/L cefotaxime and 25 mg/L kanamicine (in the case of pCB124 and pCB161 vectors using) and incubated in the light at 24º C. “Hairy” roots were subcultured every 2 weeks on the same medium.
PCR Analysis Of “Hairy” Root Cultures Polymerase Chain Reaction (PCR) method was used to detect rolB, nptII, ifn-α2b genes in established “hairy” roots or to detect virD1 gene presence (A. rhizogenes contamination in the “hairy” roots). DNA of non-transformed roots was used as the negative control.
Genome DNA was extracted from “hairy” roots according to CTAB method (20). PCR analysis of genome DNA was performed on Mastercycler personal 5332 amplfier (Eppendorf) using primers shown in the Table. The amplification conditions were as
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follows: primary denaturation – 94°C, 3 min; 30 cycles of amplification (94°C, 30 sec – 56-67°C (see tabl.1), 30-40 sec – 72°C, 30 sec); final polymerization – 72°C, 10 min.
RESULTS AND DISCUSSION Root formation both on the younger and the older leaves started in 5-6 days after
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cocultivation with bacterial suspension. There were no differences in the frequency of root formation in the case of upper or lower (younger or older) leaves used as explants. At the same time prolongation of explant cultivation on the medium without cefotaxime has led to stimulation of root growth and resulted in more than 1.5-fold increasing of root formation frequency (Fig. 1c, d) as well as in increasing of root number per explant and root length up to 7-fold and 6-fold respectively. Various factors are found to enhance A. rhizogenes-mediated transformation frequency (21). In our experiments we used neither chemicals (acetoceringone) nor physical methods (for example sonification) for Artemisia transformation and in case of genetic transformation without any additional manipulations A. tilesii ‘hairy” root culture was initiated with the frequency up to 100%.
Induced roots had typical “hairy” phenotype (Fig.1 e, f) and did not require external auxines for their growth. The transgenic nature of “hairy” roots was confirmed by PCR using rol A and nptII specific primers (Fig. 2). PCR with vir D1 primers revealed the absence of A. rhizogenes contamination (data not shown).
Variation of the duration of cultivation time on the media without antibiotics inhibiting agrobacteria growth can lead to increasing of transformation frequency either because of
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improvement of explants survival after the contact with phytopatogenic bacteria (in case of short period of cultivation with agrobacteria) or due to the time which is necessary for transfer of the bacterial Ti-plasmid genes into the plant cells. In our study we have actually observed a necessity of increasing of the cultivation time on the medium without cefotaxime for stimulation of “hairy” roots growth. Increasing of cultivation time on the
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medium without cefotaxime from one to four days after A. rhizogenes A4 wild strain transformation has led to increasing of root formation frequency from 38.9+1.92 % to 62.2+1.90 % (young leaves) and from 42.2+5.75 % to 69.0+3.77 % (old leaves). We obtained similar results using A. rhizogenes carrying pCB124 and pCB161 plasmids. Prolongation of the period of cultivation without antibiotic adding to the medium from one to four days resulted in increase of root formation frequency from 61.1+1.9 to 100% both for young and old leaves in case of pCB161 plasmid used for transformation. We have also observed in this case 4.72- fold, 1.46- fold and 2.29-fold increase of root number per explant and 6.14- fold, 1.64- fold and 1.97-fold increase of root length (respectively for using pCB124, pCB161 vectors and A. rhizogenes A4 transformation).
Established root lines differed in growth rates and biomass accumulation during cultivation on the hormone-free medium. Biomass increase of “hairy” root lines was 1525-fold higher than that of the roots of non-transgenic plants during the same period. Such effect of genetic transformation was studied earlier and is the result of transferred genes introduction to the different loci. This phenomenon opens the way to induce rapidly growing “hairy” root lines and to use them in biotechnologies for production of valuable compounds of plant origin.
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The rapid hormone-independent growth is the characteristic of “hairy” root phenotype. At the same time the use of plant growth regulators for “hairy” root in vitro cultivation can affect biochemical changes, resulting in altered growth (22-23). Addition of growth regulators can also lead to some morphological changes (24) including activation of root
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fibril growth, root thickening and root branching.
We have studied both the effect of sucrose concentration and the effect of synthetic (indole-3-butyric acid, IBA) and native growth regulators (EmistimTM, National Enterprise Interdepartmental Science and Technology Center “Agrobiotech” production) on the stimulation of A. tilesii “hairy” root culture growth. EmistimTM was chosen because earlier (25) it was shown to stimulate the growth of Cichorium intybus transgenic roots. 5-mm-long root tips from three “hairy” root lines and from roots of control nontransgenic plants were cultured on agar-solidified media supplemented with 40 g/L sucrose, 0.5 or 1.0 mg/L IBA, 5 or 10 μl/l EmistimeTM during 10 days. Maximum stimulating effect for the control roots (up to 7.95 time) was observed in case of root cultivation on the media supplemented with 0.5 or 1 mg/L IBA although cultivation on the medium with 10 μl/l EmistimeTM also led to growth stimulation (2.75-fold root mass increase).
“Hairy” roots sensitivity to growth regulators depended on root lines. Both IBA and EmistimeTM affected “hairy” root growth stimulation. Addition of 0.5 or 1.0 mg/L IBA to ½ MS medium resulted in 3.3-9.1- and 2.5-5.7-fold increasing of “hairy” root fresh
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weight respectively. EmistimeTM addition in concentration of 5 μl/L resulted in 5.46-fold biomass increase of only one transgenic root line but in the other root lines there were no statistically reliable differences in biomass increase. Favorable response to exogenous IBA application was found for A. tilesii Line 1 (0.5 mg/L IBA) and Line 3 (1.0 mg/L IBA). Root weight in these cases increased significantly compared to the growth on ½
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MS medium without growth regulators.
Increasing of sucrose content from 20 to 40 g/L has led to 1.6-5.3-fold increase of root weight in two hairy root lines but did not affect the control roots growth.
CONCLUSIONS Agrobacterium rhizogenes-mediated transformation can be used for the establishment of Artemisia tilesii “hairy” root culture. Root formation both on the younger and the older leaves started in 5-6 days after cocultivation with the bacterial suspension. The frequency of root formation was up to 100% and did not differ in the case of younger or older leaves used as explants. Prolongation of time of explant cultivation on the medium without cefotaxime has led to stimulation of root growth and resulted in more than 1.6-fold increase of root formation frequency as well as increasing of root number per explant and root length. Established root lines differed in growth rate and biomass accumulation during cultivation on the hormone-free medium. Addition of IBA or EmistimeTM growth regulator as well as increasing of sucrose content from 2% to 4% caused “hairy” root growth stimulation but the stimulating phenomenon varied for different root lines.
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REFERENCES 1.
Taylor, C.G.; Fuchs, B.; Collier, R.; Lutke, W.K. Generation of composite plants
using Agrobacterium rhizogenes. Methods Mol Biol. 2006, 343 (1), 155-167. 2.
Christey, M.C. Use of Ri-mediated transformation for production of transgenic
plants. In Vitro Cell Dev-Pl. 2001, 37 (6), 687-700.
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
3.
Christey, M.C.; Braun, R.H. Production of hairy root cultures and transgenic plants
by Agrobacterium rhizogenes-mediated transformation. Methods Mol Biol. 2005, 286, 47-60. 4.
Sevon, N.; Oksman-Caldentey, K.M. Agrobacterium rhizogenes-mediated
transformation: root culture as a source of alkaloids. Planta Med. 2002, 68 (10), 859-868. 5.
Ludwig-Müller, J.; Jahn, L.; Lippert, A.; Püschel, J.; Walter A. Improvement of
hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications. 2014, 32 (6), 1168-1179. 6.
Georgiev, M. I.; Agostini, E.; Ludwig-Müller, J.; Xu J. Genetically transformed
roots: from plant disease to biotechnological resource. Trends in Biotechnol. 2012, 30 (10), 528-537. 7.
Turi, C.E.; Shipley, P.R.; Murch, S.J. North American Artemisia species from the
subgenus Tridentatae (Sagebrush): A phytochemical, botanical and pharmacological review. Phytochem. 2014, 98, 9-26. 8.
Tang, K.; Shen, Q.; Yan, T.; Fu, X. Transgenic approach to increase artemisinin
content in Artemisia annua L. Plant Cell Rep. 2014, 33 (4), 605-615. 9.
Sujatha, G.; Zdravković-Korać, S.; Ćalić, D.; Flamini, G.; Ranjitha Kumaria, B.D.
High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia
9
vulgaris: Hairy root production and essential oil analysis. Ind Crops Prod. 2013, 44, 643652. 10.
Ali, M.; Kiani, B.H.; Mannan, A.; Ismail, T.; Mirza, B. Enhanced production of
artemisinin by hairy root cultures of Artemisia dubia. J Med Plant Res. 2012, 6 (9), 16191622.
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
11. Shuaihua, T.; Helin, W.; Mingbo, Z., Chen, Z.; Pengfei, T. Sesquiterpenes from the aerial parts of Artemisia vestita Wall. JCPS. 2013, 22 (6), 527-530. 12. Giri, A.; Ravindra, S.T.; Dhingra, V.; Lakshmi, N.M. Influence of different strains of Agrobacterium rhizogenes on induction of hairy roots and artemisin production in Artemisia annua. Curr Sci. 2001, 81 (4), 378-382. 13. Weathers, P. J.; De Jesus-Gonzalez, L.; Kim, Y.J.; Souret, F. F.; Towler, M. J. Alteration of biomass and artemisinin production in Artemisia annua hairy roots by media sterilization method and sugars. Plant Cell Rep. 2004, 23 (6), 414-418. 14. Mannan, A.; Shaheen, N.; Arshad, W.; Qureshi, R.A.; Muhammad, Zia; Bushra, Mirza. Hairy roots induction and artemisinin analysis in Artemisia dubia and Artemisia indica. Afr J Biotechnol. 2008, 7 (18), 3288–3292. 15. Griffin, D. Contributions to the ethnobotany of the Cup’it Eskimo, Nunivac Island, Alaska. J. Ethnobiol. 2001, 21 (2), Р.91-127. 16. Belokurova, V.B.; Kuchuk, N.V. In vitro bank and seed collection of wild-growing plants as a tool for plant conservation and utilization in biotechnological studies. Biotechnology and Plant Breeding Perspectives. Agrobios (International), eds. R.K.Behl and E.Arseniuk. – Babloo Offset Jodhpur, 2014, 219-232.
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17. Murashige, T; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plantarum. 1962, 15, 473-497. 18. Luchakivskaya, Yu.; Kishchenko, O.; Gerasymenko, I.; Olevinskaya, Z.; Simonenko, Yu.; Spivak M.; Kuchuk, M. High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants. Plant Cell Rep. 2011, 30 (3),
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
407-415. 19. Matvieieva, N.A.; Kishchenko, O.M.; Potrochov, A.O. Regeneration of transgenic plants from hairy roots of Cichorium intybus L. var. Foliosum Hegi. Cyt Gen. 2011, 45 (1), 277-281. 20. Draper, J.; Scott, R. The isolation of plant nucleic acids. Plant Genetic Transformation and Gene Expression: A Laboratory Manual; Blackwell Sci. Publ.: Oxford, Great Britain, 1988; 355 pp. Kumar, V.; Sharma A.; Prasad Bellur, C.N.; Bhaskar, H.G.; Ravishankar, G.A. Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment. Electronic J Biotechnol. 2006, 9 (4). DOI:10.2225/vol9-issue4-fulltext-4. 21. Bais, H.P.; Sudha, G.; George, J.; Ravishankah G.A. Influence of exogenous hormones on growth in secondary metabolite production in hairy root cultures of Chicorium intybus L. cv. lucknow local. In Vitro Cell Dev. Biol. Plant. 2001, 37 (2), 293299. 22. Bálványos, I.; Kursinszki, L.; Szõke, É. The effect of plant growth regulators on biomass formation and lobeline production of Lobelia inflata L. hairy root cultures. Plant Growth Regul. 2001, 34 (3), 339-345
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23. Farkya, S; Bisaria V.S. Exogenous hormones affecting morphology and biosynthetic potential of hairy root line (LYR2i) of Linum album. J. Biosci. Bioeng. 2008, 105 (2), 140-146 Sevon, N.; Oksman-Caldentey, K-M. Agrobacterium rhizogenesmediated transformation: root culture as a source of alkaloids. Planta Med. 2002, 68 (10), 859-868.
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24. Tsygankova, V.А.; Yemets, A.I.; Ponomarenko, S.P; Matvieieva, N.A.; Chapkevich, S.E., Kuchuk, N.V. Increase in the Synthesis of Polyfructan in the Cultures of Chicory “Hairy Roots” with Plant Natural Growth Regulators. Int J of Biomed. 2013, 3 (2), 139-144.
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TABLE 1. Primers used to confirm the presence of transgenes in A. tilesii “hairy” roots Gene
nptII
Primers
5'- cctgaatgaactccaggacgaggca-3'
Size of amplified
Annealing
fragment, b.p.
temperature
622
65°C
396
60°C
780
56°C
432
60°C
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5'- gctctagatccagagtcccgctcagaag-3' ifn-
5′-ttgatgctcctggcacag- 3′
a2b
5′-ttctgctctgacaacctc-3′
rolB
5'-atggatcccaaattgctattccttccacga-3' 5'-ttaggcttctttcttcaggtttactgcagc-3'
Vir
5’-atgtcgcaaggcagtaagccca -3’
D1
5’-ggagtctttcagcatggagcaa-3’
13
Figure 1. In vitro cultivated Artemisia tilesii plants (1a); leaves growth on MS medium without transformation (1b); “hairy” roots growth on A. tilesii leaves after A. rhizogenes– mediated transformation - one day (1c) and four days (1d) growth on the medium without cefotaxime; root formation on the leaf explant (1e); A. tilesii “hairy” root culture
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growth, two months after A. rhizogenes–mediated transformation (1f).
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Figure 2. PCR- analysis of three “hairy” root clones transformed using A. rhizogenes A4 strain (3, 8, 13), A. rhizogenes pCB124 (2, 7, 12), A. rhizogenes pCB161 (4, 9, 14); Contr - genomic DNA of non-transformed roots; -DNA - without DNA; nptII, ifn and rolB – “hairy” roots DNA analysis of the confirmation of nptII (622 bp), ifn-α2b (396 bp)
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and rolB (780 bp) genes presence; M -marker
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Preparative Biochemistry and Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb20
Artemisia Tilesii Ledeb Hairy Roots Establishment Using Agrobacterium Rhizogenes-Mediated Transformation a
a
a
a
N. A. Matvieieva , A. M. Shakhovsky , V. B. Belokurova & K. O. Drobot a
Institute of Cell Biology and genetic engineering, National Academy of Sciences of Ukraine, Kyiv, Ukraine Accepted author version posted online: 02 Apr 2015.
Click for updates To cite this article: N. A. Matvieieva, A. M. Shakhovsky, V. B. Belokurova & K. O. Drobot (2015): Artemisia Tilesii Ledeb Hairy Roots Establishment Using Agrobacterium Rhizogenes-Mediated Transformation, Preparative Biochemistry and Biotechnology, DOI: 10.1080/10826068.2015.1031393 To link to this article: http://dx.doi.org/10.1080/10826068.2015.1031393
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Artemisia tilesii Ledeb Hairy Roots Establishment Using Agrobacterium rhizogenesMediated Transformation N. A. Matvieieva1, A. M. Shakhovsky1, V. B. Belokurova1, K. O. Drobot1 1
Institute of Cell Biology and genetic engineering, National Academy of Sciences of Ukraine, Kyiv, Ukraine Corresponding author: Matvieieva Nadiia E-mail: [email protected]
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
Abstract An efficient and rapid protocol for the establishment of Artemisia tilesii “hairy” root culture is reported. Leaf explants of aseptically growing plants were co-cultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5-6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator EmistimTM on the stimulation of A.tilesii “hairy” root growth was studied. Maximum stimulating effect both for the control and transgenic roots was observed in case of root cultivation on the media supplemented with IBA - up to 7.95- and 9.1-fold biomass increase respectively. Cultivation on the medium with 10 μl/l EmistimeTM has also led to the control roots growth stimulation (up to 2.75-fold). EmistimeTM at 5 μl/l concentration led to 5.46-fold mass increase in only one “hairy” root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.
1
KEYWORDS: Artemisia tilesii Ledeb; Agrobacterium rhizogenes; “hairy” root culture; growth stimulation
INTRODUCTION Plant genetic transformation with Agrobacterium rhizogenes soil phytopathogenic
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
bacteria makes it possible to co-transform plant cells with more than one T-DNA at the same time. Agrobacterium cells can carry not only the resident A. rhizogenes Ri -plasmid containing the root locus rol genes responsible for root proliferation but also plasmids of interest carrying selective (bar, nptII and other genes) and the wide range of target genes (1-3). Using of medicinal plants for Agrobacterium rhizogenes-mediated transformation to produce compounds with medical properties in “hairy” roots is now of great interest. “Hairy” roots can be used for production of biologically active substances including secondary metabolites (4-6). They grow without addition of growth regulators and produce the metabolites of the initial plants. “Hairy” root culture are characterized also by rapid biomass increase.
Some species of Artemisia genus including A.absinthium, A.annua, A.vulgaris, A. vestita and A.dubia was studied as a natural source of secondary metabolites (7-11). Using of biotechnology approaches for antimalarial sesquiterpene artemisinin production is caused by the limited native plant resources of this genus and difficulty of its total chemical synthesis. Artemisia transgenic root culture can be an alternative for artemisinin production. Agrobacterium rhizogenes-mediated transformation method was used for A. annua (12, 13), A. dubia, A. indica (14), A. vulgaris (9) ”hairy” roots induction. At the
2
same time up to now any investigation was not carried out to establish A. tilesii “hairy” root culture. A. tilesii known as Aleutian mugwort has the high ability for adaptation to abiotic stress conditions. These plants also are characterized by antirheumatic, disinfectant, deodorant, anti-tumor effect and used in Alaska traditional medicine (15).
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
Induction and studying of A. tilesii “hairy” root culture growth was the aim of our work.
EXPERIMENTAL Plant Material And Bacteria Strains Used For Transformation Artemisia tilesii Ledeb seeds were used to induce in vitro culture. The seeds were obtained from the Polar-Alpine Botanical Garden (Kirovsk, Hibines, Russia) in 1997 and stored in the Seed Bank of the Institute of Cell Biology and Genetic Engineering (16). Artemisia tilesii plants cultured in vitro (Fig.1a) via micropropagation on hormone-free MS basal medium (17) were used for genetic transformation experiments.
Agrobacterium rhizogenes A4 wild strain and the bacteria carrying the binary vectors pCB124 (18) and pCB161 (19) were grown in LB liquid culture medium containing 10 g/l casein hydrolyzed, 5 g/l yeast extract, 10 g/l NaCl, 100 mg/l carbenicillin, 50 mg/l rifampicin, pH 7.0 overnight at 28°C in the dark on shaker (220 rpm). A.rhizogenes A4 strain was cultivated in LB liquid culture medium without antibiotics.
“Hairy” Root Induction Slightly sliced upper and lower leaf explants isolated from 30-days-old in vitro grown plants were co-cultured with A. rhizogenes suspension during 30 min, transferred to the
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agar-solidified 1/2MS medium (MS medium with twice reduced macrosalts content) in Petri dishes and cultured for 48 hrs in the dark at 28º C. Then they were transferred to the agar-solidified 1/2MS medium (20 g/L sucrose, pH 5.7) supplemented with 600 mg/L cefotaxime for agrobacteria elimination. Thirty explants were used in each experiment (the experiments were performed in triplicate). Root induction frequency was measured
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as the ratio of the number of explants with induced roots to the total number of explants used for transformation.
Initiated “hairy” roots were transferred on 1/2 MS medium supplemented with 2 % sucrose, 0.7% agar, 600 mg/L cefotaxime and 25 mg/L kanamicine (in the case of pCB124 and pCB161 vectors using) and incubated in the light at 24º C. “Hairy” roots were subcultured every 2 weeks on the same medium.
PCR Analysis Of “Hairy” Root Cultures Polymerase Chain Reaction (PCR) method was used to detect rolB, nptII, ifn-α2b genes in established “hairy” roots or to detect virD1 gene presence (A. rhizogenes contamination in the “hairy” roots). DNA of non-transformed roots was used as the negative control.
Genome DNA was extracted from “hairy” roots according to CTAB method (20). PCR analysis of genome DNA was performed on Mastercycler personal 5332 amplfier (Eppendorf) using primers shown in the Table. The amplification conditions were as
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follows: primary denaturation – 94°C, 3 min; 30 cycles of amplification (94°C, 30 sec – 56-67°C (see tabl.1), 30-40 sec – 72°C, 30 sec); final polymerization – 72°C, 10 min.
RESULTS AND DISCUSSION Root formation both on the younger and the older leaves started in 5-6 days after
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cocultivation with bacterial suspension. There were no differences in the frequency of root formation in the case of upper or lower (younger or older) leaves used as explants. At the same time prolongation of explant cultivation on the medium without cefotaxime has led to stimulation of root growth and resulted in more than 1.5-fold increasing of root formation frequency (Fig. 1c, d) as well as in increasing of root number per explant and root length up to 7-fold and 6-fold respectively. Various factors are found to enhance A. rhizogenes-mediated transformation frequency (21). In our experiments we used neither chemicals (acetoceringone) nor physical methods (for example sonification) for Artemisia transformation and in case of genetic transformation without any additional manipulations A. tilesii ‘hairy” root culture was initiated with the frequency up to 100%.
Induced roots had typical “hairy” phenotype (Fig.1 e, f) and did not require external auxines for their growth. The transgenic nature of “hairy” roots was confirmed by PCR using rol A and nptII specific primers (Fig. 2). PCR with vir D1 primers revealed the absence of A. rhizogenes contamination (data not shown).
Variation of the duration of cultivation time on the media without antibiotics inhibiting agrobacteria growth can lead to increasing of transformation frequency either because of
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improvement of explants survival after the contact with phytopatogenic bacteria (in case of short period of cultivation with agrobacteria) or due to the time which is necessary for transfer of the bacterial Ti-plasmid genes into the plant cells. In our study we have actually observed a necessity of increasing of the cultivation time on the medium without cefotaxime for stimulation of “hairy” roots growth. Increasing of cultivation time on the
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medium without cefotaxime from one to four days after A. rhizogenes A4 wild strain transformation has led to increasing of root formation frequency from 38.9+1.92 % to 62.2+1.90 % (young leaves) and from 42.2+5.75 % to 69.0+3.77 % (old leaves). We obtained similar results using A. rhizogenes carrying pCB124 and pCB161 plasmids. Prolongation of the period of cultivation without antibiotic adding to the medium from one to four days resulted in increase of root formation frequency from 61.1+1.9 to 100% both for young and old leaves in case of pCB161 plasmid used for transformation. We have also observed in this case 4.72- fold, 1.46- fold and 2.29-fold increase of root number per explant and 6.14- fold, 1.64- fold and 1.97-fold increase of root length (respectively for using pCB124, pCB161 vectors and A. rhizogenes A4 transformation).
Established root lines differed in growth rates and biomass accumulation during cultivation on the hormone-free medium. Biomass increase of “hairy” root lines was 1525-fold higher than that of the roots of non-transgenic plants during the same period. Such effect of genetic transformation was studied earlier and is the result of transferred genes introduction to the different loci. This phenomenon opens the way to induce rapidly growing “hairy” root lines and to use them in biotechnologies for production of valuable compounds of plant origin.
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The rapid hormone-independent growth is the characteristic of “hairy” root phenotype. At the same time the use of plant growth regulators for “hairy” root in vitro cultivation can affect biochemical changes, resulting in altered growth (22-23). Addition of growth regulators can also lead to some morphological changes (24) including activation of root
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fibril growth, root thickening and root branching.
We have studied both the effect of sucrose concentration and the effect of synthetic (indole-3-butyric acid, IBA) and native growth regulators (EmistimTM, National Enterprise Interdepartmental Science and Technology Center “Agrobiotech” production) on the stimulation of A. tilesii “hairy” root culture growth. EmistimTM was chosen because earlier (25) it was shown to stimulate the growth of Cichorium intybus transgenic roots. 5-mm-long root tips from three “hairy” root lines and from roots of control nontransgenic plants were cultured on agar-solidified media supplemented with 40 g/L sucrose, 0.5 or 1.0 mg/L IBA, 5 or 10 μl/l EmistimeTM during 10 days. Maximum stimulating effect for the control roots (up to 7.95 time) was observed in case of root cultivation on the media supplemented with 0.5 or 1 mg/L IBA although cultivation on the medium with 10 μl/l EmistimeTM also led to growth stimulation (2.75-fold root mass increase).
“Hairy” roots sensitivity to growth regulators depended on root lines. Both IBA and EmistimeTM affected “hairy” root growth stimulation. Addition of 0.5 or 1.0 mg/L IBA to ½ MS medium resulted in 3.3-9.1- and 2.5-5.7-fold increasing of “hairy” root fresh
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weight respectively. EmistimeTM addition in concentration of 5 μl/L resulted in 5.46-fold biomass increase of only one transgenic root line but in the other root lines there were no statistically reliable differences in biomass increase. Favorable response to exogenous IBA application was found for A. tilesii Line 1 (0.5 mg/L IBA) and Line 3 (1.0 mg/L IBA). Root weight in these cases increased significantly compared to the growth on ½
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MS medium without growth regulators.
Increasing of sucrose content from 20 to 40 g/L has led to 1.6-5.3-fold increase of root weight in two hairy root lines but did not affect the control roots growth.
CONCLUSIONS Agrobacterium rhizogenes-mediated transformation can be used for the establishment of Artemisia tilesii “hairy” root culture. Root formation both on the younger and the older leaves started in 5-6 days after cocultivation with the bacterial suspension. The frequency of root formation was up to 100% and did not differ in the case of younger or older leaves used as explants. Prolongation of time of explant cultivation on the medium without cefotaxime has led to stimulation of root growth and resulted in more than 1.6-fold increase of root formation frequency as well as increasing of root number per explant and root length. Established root lines differed in growth rate and biomass accumulation during cultivation on the hormone-free medium. Addition of IBA or EmistimeTM growth regulator as well as increasing of sucrose content from 2% to 4% caused “hairy” root growth stimulation but the stimulating phenomenon varied for different root lines.
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REFERENCES 1.
Taylor, C.G.; Fuchs, B.; Collier, R.; Lutke, W.K. Generation of composite plants
using Agrobacterium rhizogenes. Methods Mol Biol. 2006, 343 (1), 155-167. 2.
Christey, M.C. Use of Ri-mediated transformation for production of transgenic
plants. In Vitro Cell Dev-Pl. 2001, 37 (6), 687-700.
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
3.
Christey, M.C.; Braun, R.H. Production of hairy root cultures and transgenic plants
by Agrobacterium rhizogenes-mediated transformation. Methods Mol Biol. 2005, 286, 47-60. 4.
Sevon, N.; Oksman-Caldentey, K.M. Agrobacterium rhizogenes-mediated
transformation: root culture as a source of alkaloids. Planta Med. 2002, 68 (10), 859-868. 5.
Ludwig-Müller, J.; Jahn, L.; Lippert, A.; Püschel, J.; Walter A. Improvement of
hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications. 2014, 32 (6), 1168-1179. 6.
Georgiev, M. I.; Agostini, E.; Ludwig-Müller, J.; Xu J. Genetically transformed
roots: from plant disease to biotechnological resource. Trends in Biotechnol. 2012, 30 (10), 528-537. 7.
Turi, C.E.; Shipley, P.R.; Murch, S.J. North American Artemisia species from the
subgenus Tridentatae (Sagebrush): A phytochemical, botanical and pharmacological review. Phytochem. 2014, 98, 9-26. 8.
Tang, K.; Shen, Q.; Yan, T.; Fu, X. Transgenic approach to increase artemisinin
content in Artemisia annua L. Plant Cell Rep. 2014, 33 (4), 605-615. 9.
Sujatha, G.; Zdravković-Korać, S.; Ćalić, D.; Flamini, G.; Ranjitha Kumaria, B.D.
High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia
9
vulgaris: Hairy root production and essential oil analysis. Ind Crops Prod. 2013, 44, 643652. 10.
Ali, M.; Kiani, B.H.; Mannan, A.; Ismail, T.; Mirza, B. Enhanced production of
artemisinin by hairy root cultures of Artemisia dubia. J Med Plant Res. 2012, 6 (9), 16191622.
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
11. Shuaihua, T.; Helin, W.; Mingbo, Z., Chen, Z.; Pengfei, T. Sesquiterpenes from the aerial parts of Artemisia vestita Wall. JCPS. 2013, 22 (6), 527-530. 12. Giri, A.; Ravindra, S.T.; Dhingra, V.; Lakshmi, N.M. Influence of different strains of Agrobacterium rhizogenes on induction of hairy roots and artemisin production in Artemisia annua. Curr Sci. 2001, 81 (4), 378-382. 13. Weathers, P. J.; De Jesus-Gonzalez, L.; Kim, Y.J.; Souret, F. F.; Towler, M. J. Alteration of biomass and artemisinin production in Artemisia annua hairy roots by media sterilization method and sugars. Plant Cell Rep. 2004, 23 (6), 414-418. 14. Mannan, A.; Shaheen, N.; Arshad, W.; Qureshi, R.A.; Muhammad, Zia; Bushra, Mirza. Hairy roots induction and artemisinin analysis in Artemisia dubia and Artemisia indica. Afr J Biotechnol. 2008, 7 (18), 3288–3292. 15. Griffin, D. Contributions to the ethnobotany of the Cup’it Eskimo, Nunivac Island, Alaska. J. Ethnobiol. 2001, 21 (2), Р.91-127. 16. Belokurova, V.B.; Kuchuk, N.V. In vitro bank and seed collection of wild-growing plants as a tool for plant conservation and utilization in biotechnological studies. Biotechnology and Plant Breeding Perspectives. Agrobios (International), eds. R.K.Behl and E.Arseniuk. – Babloo Offset Jodhpur, 2014, 219-232.
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17. Murashige, T; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plantarum. 1962, 15, 473-497. 18. Luchakivskaya, Yu.; Kishchenko, O.; Gerasymenko, I.; Olevinskaya, Z.; Simonenko, Yu.; Spivak M.; Kuchuk, M. High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants. Plant Cell Rep. 2011, 30 (3),
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
407-415. 19. Matvieieva, N.A.; Kishchenko, O.M.; Potrochov, A.O. Regeneration of transgenic plants from hairy roots of Cichorium intybus L. var. Foliosum Hegi. Cyt Gen. 2011, 45 (1), 277-281. 20. Draper, J.; Scott, R. The isolation of plant nucleic acids. Plant Genetic Transformation and Gene Expression: A Laboratory Manual; Blackwell Sci. Publ.: Oxford, Great Britain, 1988; 355 pp. Kumar, V.; Sharma A.; Prasad Bellur, C.N.; Bhaskar, H.G.; Ravishankar, G.A. Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment. Electronic J Biotechnol. 2006, 9 (4). DOI:10.2225/vol9-issue4-fulltext-4. 21. Bais, H.P.; Sudha, G.; George, J.; Ravishankah G.A. Influence of exogenous hormones on growth in secondary metabolite production in hairy root cultures of Chicorium intybus L. cv. lucknow local. In Vitro Cell Dev. Biol. Plant. 2001, 37 (2), 293299. 22. Bálványos, I.; Kursinszki, L.; Szõke, É. The effect of plant growth regulators on biomass formation and lobeline production of Lobelia inflata L. hairy root cultures. Plant Growth Regul. 2001, 34 (3), 339-345
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23. Farkya, S; Bisaria V.S. Exogenous hormones affecting morphology and biosynthetic potential of hairy root line (LYR2i) of Linum album. J. Biosci. Bioeng. 2008, 105 (2), 140-146 Sevon, N.; Oksman-Caldentey, K-M. Agrobacterium rhizogenesmediated transformation: root culture as a source of alkaloids. Planta Med. 2002, 68 (10), 859-868.
Downloaded by [University of Nebraska, Lincoln] at 19:20 06 April 2015
24. Tsygankova, V.А.; Yemets, A.I.; Ponomarenko, S.P; Matvieieva, N.A.; Chapkevich, S.E., Kuchuk, N.V. Increase in the Synthesis of Polyfructan in the Cultures of Chicory “Hairy Roots” with Plant Natural Growth Regulators. Int J of Biomed. 2013, 3 (2), 139-144.
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TABLE 1. Primers used to confirm the presence of transgenes in A. tilesii “hairy” roots Gene
nptII
Primers
5'- cctgaatgaactccaggacgaggca-3'
Size of amplified
Annealing
fragment, b.p.
temperature
622
65°C
396
60°C
780
56°C
432
60°C
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5'- gctctagatccagagtcccgctcagaag-3' ifn-
5′-ttgatgctcctggcacag- 3′
a2b
5′-ttctgctctgacaacctc-3′
rolB
5'-atggatcccaaattgctattccttccacga-3' 5'-ttaggcttctttcttcaggtttactgcagc-3'
Vir
5’-atgtcgcaaggcagtaagccca -3’
D1
5’-ggagtctttcagcatggagcaa-3’
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Figure 1. In vitro cultivated Artemisia tilesii plants (1a); leaves growth on MS medium without transformation (1b); “hairy” roots growth on A. tilesii leaves after A. rhizogenes– mediated transformation - one day (1c) and four days (1d) growth on the medium without cefotaxime; root formation on the leaf explant (1e); A. tilesii “hairy” root culture
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growth, two months after A. rhizogenes–mediated transformation (1f).
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Figure 2. PCR- analysis of three “hairy” root clones transformed using A. rhizogenes A4 strain (3, 8, 13), A. rhizogenes pCB124 (2, 7, 12), A. rhizogenes pCB161 (4, 9, 14); Contr - genomic DNA of non-transformed roots; -DNA - without DNA; nptII, ifn and rolB – “hairy” roots DNA analysis of the confirmation of nptII (622 bp), ifn-α2b (396 bp)
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and rolB (780 bp) genes presence; M -marker
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