Abstract
The introgression of rol-genes of rhizogenic Agrobacterium into the plant genome induces changes in plant phenotype and physiology. However, only limited experience with this technique is available for woody ornamentals. To induce new variation within the Escallonia genus, several species were co-cultivated with rhizogenic Agrobacterium strains. Co-cultivation of three rhizogenic Agrobacterium strains (Arqua1, LMG 63 and MAFF210266) with four Escallonia species (E. illinita, E. myrtoidea, E. rosea, and E. rubra), resulted in hairy roots production with a varying efficiency. Co-cultivation of E. rubra with MAFF210266, and E. myrtoidea with LMG63 did not yield hairy roots, while co-cultivation of E. rubra leaves with LMG63 was most successful for hairy root production (up to 80.6%). In addition, the efficiency of hairy root induction depended on the explant type (leaves or nodal sections). The presence of inserted rol-genes and aux-genes in hairy roots was molecularly confirmed using qPCR. Few shoots regenerated from hairy roots, but regeneration needs to be optimized for efficient implementation of rol-genes introgression in Escallonia breeding.
Key Message
This research provides a protocol for the production of hairy roots with rol-genes inserted after co-cultivation of several species of Escallonia with rhizogenic Agrobacterium strains.
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References
Alpizar E, Dechamp E, Espeout S et al (2006) Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots. Plant Cell Rep 25:959–967. https://doi.org/10.1007/s00299-006-0159-9
Amoo SO, Finnie JF, Van Staden J (2011) The role of meta-topolins in alleviating micropropagation problems. Plant Growth Regul 63:197–206. https://doi.org/10.1007/s10725-010-9504-7
Bean WJ, Murray J (1989) Trees and shrubs hardy in the British Isles. Butler and Tanner Ltd., London
Bettini PP, Santangelo E, Baraldi R et al (2016) Agrobacterium rhizogenes rolA gene promotes tolerance to Fusarium oxysporum f. sp. lycopersici in transgenic tomato plants (Solanum lycopersicum L.). J Plant Biochem Biotechnol 25:225–233. https://doi.org/10.1007/s13562-015-0328-4
Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotechnol Adv 26:318–324. https://doi.org/10.1016/j.biotechadv.2008.03.001
Camilleri C, Jouanin L (1991) The TR-DNA region carrying the auxin synthesis genes of the Agrobacterium rhizogenes agropine-type plasmid pRiA4: nucleotide sequence analysis and introduction into tobacco plants. Mol Plant-Microbe Interact 4:155–162
Cheng T, Xu C, Lei L et al (2016) Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity. Mol Ecol Resour 16:138–149. https://doi.org/10.1111/1755-0998.12438
Chilton M-D, Tepfer DA, Petit A et al (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295:432–434. https://doi.org/10.1038/295432a0
Choi PS, Kim YD, Choi KM et al (2004) Plant regeneration from hairy-root cultures transformed by infection with Agrobacterium rhizogenes in Catharanthus roseus. Plant Cell Rep 22:828–831. https://doi.org/10.1007/s00299-004-0765-3
Christensen B, Müller R (2009) Kalanchoe blossfeldiana transformed with rol genes exhibits improved postharvest performance and increased ethylene tolerance. Postharvest Biol Technol 51:399–406. https://doi.org/10.1016/j.postharvbio.2008.08.010
Christensen B, Sriskandarajah S, Müller R (2009) Transformation of Hibiscus rosa-sinensis L. by Agrobacterium rhizogenes. J Hortic Sci Biotechnol 84:204–208. https://doi.org/10.1080/14620316.2009.11512505
Denaeghel HER, Van Laere K, Leus L et al (2018) The variable effect of polyploidization on the phenotype in Escallonia. Front Plant Sci 9:1–17. https://doi.org/10.3389/fpls.2018.00354
Desmet S, De Keyser E, Van Vaerenbergh J et al (2019) Differential efficiency of wild type rhizogenic strains for rol gene transformation of plants. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-019-10003-0
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus (Madison) 12:39–40
Georgiev MI, Agostini E, Ludwig-Müller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol 30:528–537. https://doi.org/10.1016/j.tibtech.2012.07.001
Haas JH, Moore LW, Ream W, Manulis S (1995) Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:2879–2884
Hegelund JN, Lauridsen UB, Wallström SV et al (2017) Transformation of Campanula by wild type Agrobacterium rhizogenes. Euphytica. https://doi.org/10.1007/s10681-017-1845-0
He-Ping S, Yong-Yue L, Tie-Shan S, Eric TPK (2011) Induction of hairy roots and plant regeneration from the medicinal plant Pogostemon Cablin. Plant Cell Tissue Organ Cult 107:251–260. https://doi.org/10.1007/s11240-011-9976-9
Nurseries H (1991) The Hillier manual of trees and shrubs, 6th edn. David and Charles, Winchester
Hoffman M, Ravesloot M (1998) Winterhardheid van boomkwekerijgewassen. Prakt. Plant Omgeving Sect, Bomen
Huffman GA, White FF, Gordon MP, Nester EW (1984) Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids. J Bacteriol 157:269–276
Jouanin L, Guerche P, Pamboukdjian N et al (1987) Structure of T-DNA in plants regenerated from roots transformed by Agrobacterium rhizogenes strain A4. Mol Gen Genet 206:387–392
Kang HJ, Anbazhagan VR, You XL et al (2006) Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots. Plant Cell Tissue Organ Cult 85:187–196. https://doi.org/10.1007/s11240-005-9070-2
Khan SA, Verma P, Banerjee S et al (2017) Pyrethrin accumulation in elicited hairy root cultures of Chrysanthemum cinerariaefolium. Plant Growth Regul 81:365–376. https://doi.org/10.1007/s10725-016-0213-8
Kim YS, Kim YK, Xu H et al (2012) Improvement of ornamental characteristics in Rehmannia elata through Agrobacterium rhizogenes-mediated transformation. Plant Omics 5:376–380
Koshita Y, Nakamura Y, Kobayashi S, Morinaga K (2002) Introduction of the rolC gene into the genome of the Japanese persimmon causes dwarfism. J Jpn Soc Hortic Sci 71:529–531. https://doi.org/10.2503/jjshs.71.529
Krüsmann G (1960) Handbuch der Laubgehölze. Berlin und Hamburg, Berlin
Lacroix B, Citovsky V (2016) Transfer of DNA from bacteria to eukaryotes. MBio 7:1–9. https://doi.org/10.1128/mbio.00863-16
Leroy T, Alpizar E, Dufour M, Etienne H (2006) Coffee (Coffea sp.). In: Wang K (ed) Agrobacterium protocols, vol 2. Humana Press, Totowa, pp 191–208
Lütken H, Jensen EB, Wallström SV et al (2012a) Development and evaluation of a non-gmo breeding technique exemplified by Kalanchoë. Acta Hortic 961:51–58. https://doi.org/10.17660/ActaHortic.2012.961.3
Lütken H, Wallström SV, Jensen EB et al (2012) Inheritance of rol-genes from Agrobacterium rhizogenes through two generations in Kalanchoë. Euphytica 188:397–407. https://doi.org/10.1007/s10681-012-0701-5
Majumdar S, Garai S, Jha S (2011) Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants. Plant Cell Rep 30:941–954. https://doi.org/10.1007/s00299-011-1035-9
Maurel C, Leblanc N, Barbier-Brygoo H et al (1994) Alterations of auxin perception in rolB-transformed tobacco protoplasts (time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin). Plant Physiol 105:1209–1215. https://doi.org/10.1104/pp.105.4.1209
Mauro ML, Costantino P, Bettini PP (2017) The never ending story of rol genes: a century after. Plant Cell Tissue Organ Cult 131:201–212. https://doi.org/10.1007/s11240-017-1277-5
Mehrotra S, Goel MK, Rahman LU, Kukreja AK (2013) Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina. Plant Cell Tissue Organ Cult 114:31–38. https://doi.org/10.1007/s11240-013-0302-6
Motte H, Vereecke D, Geelen D, Werbrouck S (2014) The molecular path to in vitro shoot regeneration. Biotechnol Adv 32:107–121. https://doi.org/10.1016/j.biotechadv.2013.12.002
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Neb D, Das A, Hintelmann A, Nehls U (2017) Composite poplars: a novel tool for ectomycorrhizal research. Plant Cell Rep 36:1959–1970. https://doi.org/10.1007/s00299-017-2212-2
Panda BM, Mehta UJ, Hazra S (2017) Optimizing culture conditions for establishment of hairy root culture of Semecarpus anacardium L. 3 Biotech 7:21. https://doi.org/10.1007/s13205-017-0608-x
Petrova M, Zayova E, Vlahova M (2013) Induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes. Cent Eur J Biol 8:470–479. https://doi.org/10.2478/s11535-013-0157-6
Piispanen R, Aronen T, Chen X et al (2003) Silver birch (Betula pendula) plants with aux and rol genes show consistent changes in morphology, xylem structure and chemistry. Tree Physiol 23:721–733. https://doi.org/10.1093/treephys/23.11.721
Pitzschke A (2013) Agrobacterium infection and plant defense—transformation success hangs by a thread. Front Plant Sci 4:1–12. https://doi.org/10.3389/fpls.2013.00519
Porter JR, Flores H (1991) Host range and implications of plant infection by Agrobacterium rhizogenes. Crit Rev Plant Sci 10:387–421. https://doi.org/10.1080/07352689109382318
Prabhu SA, Ndlovu B, Engelbrecht J, Van Den Berg N (2017) Generation of composite Persea americana (Mill.) (Avocado) plants: a proof-of-concept-study. PLoS ONE 12:1–22. https://doi.org/10.1371/journal.pone.0185896
Rastogi S, Rizvi SMH, Singh RP, Dwivedi UN (2008) In vitro regeneration of Leucaena leucocephala by organogenesis and somatic embryogenesis. Biol Plant 52:743–748. https://doi.org/10.1007/s10535-008-0144-y
Royal Horticultural Society (2018) Award of Garden Merit Plants November 2018—Ornamentals. https://www.rhs.org.uk/plants/pdfs/agm-lists/agm-ornamentals.pdf. Accessed 12 Oct 2019
Rugini E, Pellegrineschi A, Mencuccini M, Mariotti D (1991) Increase of rooting ability in the woody species kiwi (Actinidia deliciosa A. Chev.) by transformation with Agrobacterium rhizogenes rol genes. Plant Cell Rep 10:291–295. https://doi.org/10.1007/BF00193144
Subotić A, Budimir S, Grubišić D, Momčilović I (2003) Direct regeneration of shoots from hairy root cultures of Centaurium erythraea inoculated with Agrobacterium rhizogenes. Biol Plant 47:617–619. https://doi.org/10.1023/B:BIOP.0000041074.81033.3a
Tepfer D (1984) Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype. Cell 37:959–967. https://doi.org/10.1016/0092-8674(84)90430-6
Trypsteen M, Van Lijsebettens M, Van Severen R, Van Montagu M (1991) Agrobacterium rhizogenes-mediated transformation of Echinacea purpurea. Plant Cell Rep 10:85–89. https://doi.org/10.1007/BF00236463
Tsuro M, Ikedo H (2011) Changes in morphological phenotypes and essential oil components in lavandin (Lavandula × intermedia Emeric ex Loisel.) transformed with wild-type strains of Agrobacterium rhizogenes. Sci Hortic (Amst) 130:647–652. https://doi.org/10.1016/j.scienta.2011.08.011
Vladimirov IA, Matveeva TV, Lutova LA (2015) Opine biosynthesis and catabolism genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Russ J Genet 51:121–129. https://doi.org/10.1134/S1022795415020167
White FF, Taylor BH, Huffman GA et al (1985) Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol 164:33–44
Wu J, Wang Y, Zhang LX et al (2012) High-efficiency regeneration of Agrobacterium rhizogenes-induced hairy root in apple rootstock Malus baccata (L.) Borkh. Plant Cell Tissue Organ Cult 111:183–189. https://doi.org/10.1007/s11240-012-0182-1
Zdravković-Korać S, Muhovski Y, Druart P et al (2004) Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastanum L. and the regeneration of transformed plants. Plant Cell Rep 22:698–704. https://doi.org/10.1007/s00299-004-0756-4
Zuker A, Tzfira T, Scovel G et al (2001) RolC-transgenic carnation with improved horticultural traits: quantitative and qualitative analyses of greenhouse-grown plants. J Am Soc Hortic Sci 126:13–18. https://doi.org/10.21273/JASHS.126.1.13
Acknowledgements
The authors want to thank Laurence Desmet and Magali Losschaert for their technical support with the DNA-analysis and PCRs and Kristien Janssens for her assistance with the co-cultivation.
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This study was funded by BestSelect CVBA.
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Study conception and design: HD, KVL, ED, EDK, TE, SD, JVH, MVL. Acquisition of data: HD, SD. Analysis and interpretation of data: HD, KVL, SD, EDK, MVL. Drafting of manuscript: HD, SD, KVL. Critical revision: TE, ED, EDK, SD, JVH, MVL.
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Communicated by Wenwu Guo.
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Denaeghel, H.E.R., Desmet, S., De Keyser, E. et al. Introgression of rol genes from rhizogenic Agrobacterium strains into Escallonia spp.. Plant Cell Tiss Organ Cult 140, 403–414 (2020). https://doi.org/10.1007/s11240-019-01736-x
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DOI: https://doi.org/10.1007/s11240-019-01736-x