Abstract
A complete protocol of in vitro selection and greenhouse screening for glyphosate-tolerant variants in manilagrass (Zoysia matrella [L.] Merr) was established in this study. Newly subcultured calli of more than 5 years’ old were transferred to selection medium containing 2 mM glyphosate. After two rounds of selection, 220 calli survived out of 840 and were transferred to regeneration medium without glyphosate. Regenerated plantlets were then transferred to regeneration medium containing 0.5 mM glyphosate to select tolerant plantlets. After 1-month growth, there were plantlets remained green and new shoots formed beside or on discolored explants. These surviving organisms were then transferred to fresh regeneration medium for further growth. Fully developed plantlets were transferred to a green house and then subjected to greenhouse screening by foliar spraying with 0.05 % glyphosate solution. Six glyphosate-tolerant plantlets, TP1-TP6, were obtained and proliferated for determination of sod-tolerance using morphological and physiological measurements. Fourteen days after foliar application with 0.1 % glyphosate, only TP5 showed enhanced sod-tolerance. The dark green color index value of TP5 was significantly higher than CK2, demonstrating that TP5 suffered less injury from glyphosate than CK2. Different physiological characters were also observed in CK1, CK2 and TP5. Significantly higher chlorophyll a content and catalase activity were observed in TP5 than in CK1. Fourteen days after treatment (DAT), the ion leakage, proline content and ascorbate peroxidase activity of CK2 and TP5 increased significantly, but the ion leakage of TP5 was significantly lower than that of CK2. The guaiacol peroxidase activity of TP5 increased significantly 14 DAT, and was significantly higher than that of CK1 and CK2. No change in shikimate content was observed in CK2 or TP5 14 DAT.
Similar content being viewed by others
Abbreviations
- MS:
-
Murashige and Skoog
- 2,4-D:
-
2,4-Dichlorophenoxyacetic acid
- BA:
-
6-Benzyladenine
- DGCI:
-
Dark green colour index
- CHL:
-
Chlorophyll
- SOD:
-
Superoxide dismutase
- CAT:
-
Catalase
- G-POD:
-
Guaiacol peroxidase
- APX:
-
Ascorbate peroxidase
- ASA:
-
Ascorbate
- ROS:
-
Reactive oxygen species
References
Ahsan N, Lee DG, Lee KW, Alam I, Lee SH, Bahk JD, Lee BH (2008) Glyphosate-induced oxidative stress in rice leaves revealed by proteomic approach. Plant Physio Bioch 46(12):1062–1070
Baerson SR, Rodriguez DJ, Biest NA, Tran M, You J, Kreuger RW, Dill GM, Pratley JE, Gruys KJ (2002) Investigating the mechanism of glyphosate resistance in rigid ryegrass (Lolium rigidum). Weed Sci 50:721–730
Bowler C, van Montagu M, Inze D (1992) Superoxide dismutase and stress tolerance. Annu Rev Plant Physiol Plant Mol Biol 43:83–116
Brosnan JT, Breeden GK, Mueller TC (2012) A glyphosate-resistant biotype of annual bluegrass in Tennessee. Weed Sci 60(1):97–100
Chai ML, Lee JM, Park MH, Kim DH (2001) In vitro selection for large patch resistance in zoysiagrass. J. Korean Soc Hortic Sci 42(3):249–253
Chai ML, Jia YF, Chen S, Gao ZS, Wang HF, Liu LL, Wang PJ, Hou DQ (2011) Callus induction, plant regeneration, and long-term maintenance of embryogenic cultures in Zoysia matrella [L.] Merr. Plant Cell Tiss Organ Cult 104:187–192
Chen S, Chai ML, Jia YF, Gao ZS, Zhang L, Gu MX (2011) In vitro selection of salt tolerant variants following 60Co gamma irradiation of long-term callus cultures of Zoysia matrella [L.] Merr. Plant Cell Tiss Org Cult 107:493–500
Croughan SS, Quisenberry SS, Eichhorn MM, Colyer PD, Brown TF (1994) Registration of Brazos-R3 bermudagrass germplasm. Crop Sci 34:542
Demir Y (2000) Growth and proline content of germinating wheat genotypes under ultraviolet light. Turk J Bot 24:67–70
Douglas LH, Wayne AP, Paul LR (2010) Obtaining sethoxydim resistance in seashore paspalum. Crop Sci 50:2632–2640
Geiger DR, Bestman HD (1990) Self-limitation of herbicide mobility by phytotoxic action. Weed Sci 38:324–329
Goldman JJ, Hanna WW, Fleming GH, Ozias-Akins P (2004) Ploidy variation among herbicide-resistant bermudagrass plants of cv. TifEagle transformed with the bar gene. Plant Cell Rep 22:553–560
Haslam E (1993) Shikimic acid: metabolism and metabolites. Wiley, New York
Hollander H, Amrhein N (1980) The site of action of the inhibition of the shikimate pathway by glyphosate. I. Inhibition by glyphosate of phenylpropanoid synthesis in buckwheat (Fagopyrum esculentum Moench). Plant Physiol 66:823–829
Karcher DE, Richardson MD (2003) Quantifying turfgrass color using digital image analysis. Crop Sci 43:943–951
Karcher DE, Richardson MD (2005) Batch analysis of digital images to evaluate turfgrass characteristics. Crop Sci 45:1536–1539
Lee SH, Ahsan N, Lee KW, Kim DH, Lee DG, Kwak SS, Kwon SY, Kim TH, Lee BH (2007) Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses. J Plant Physiol 164(12):1626–1638
Lee HJ, Lee GJ, Kim DS, Kim JB, Ku JH, Kang SY (2008) Selection and physiological characterization of glyphosate-tolerant zoysiagrass mutants derived from a gamma ray irradiation. Kor J Hort Sci Technol 26(4):454–463
Lee KW, Kim KY, Kim KH, Lee BH, Kim JS, Lee SH (2011) Development of antibiotic marker-free creeping bentgrass resistance against herbicides. Acta Biochim Biophys Sin 43:13–18
Libik-Konieczny M, Konieczny R, Surówka E, Ślesak I, Michalec Z, Rozpądek P, Miszalski Z (2012) Pathways of ROS homeostasis regulation in Mesembryanthemum crystallinum L. calli exhibiting differences in rhizogenesis. Plant Cell Tiss Organ Cult. doi:10.1007/s11240-012-0136-7
Lokhande VH, Nikam TD, Penna S (1999) Biochemical, physiological and growth changes in response to salinity in callus cultures of Sesuvium portulacastrum L. Plant Cell Tiss Org Cult 102:17–25
Lokhande VH, Nikam TD, Patade VY, Ahire ML, Suprasanna P (2011) Effects of optimal and supra-optimal salinity stress on antioxidative defence, osmolytes and in vitro growth responses in Sesuvium portulacastrum L. Plant Cell Tiss Organ Cult 104:41–49
Lu S, Guo Z, Chen Y (2003) Preliminary study on tissue culture of bermudagrass and its dwarf somaclonal variant. Acta Hort Sin 30:482–484
Lu S, Wang Z, Peng X, Guo Z, Zhang G, Han L (2006) An efficient callus suspension culture system for triploid bermudagrass (Cynodon transvaalensis × C. dactylon) and somaclonal variations. Plant Cell, Tissue Organ Cult 87:77–84
Lu S, Peng X, Guo Z, Zhang G, Wang Z, Wang C, Pang C, Fan Z, Wang J (2007) In vitro selection of salinity tolerant variants from triploid bermudagrass (Cynodon transvaalensis × C. dactylon) and their physiological responses to salt and drought stress. Plant Cell Rep 26:1413–1420
Luo XY, Matsumoto H (2002) Susceptibility of a broad-leaved weed, Acanthospermum hispidum, to the grass herbicide fluazifop-butyl. Weed Biol Manage 2:98–103
Mueller TC, Massey JH, Hayes RM, Main CL, Stewart N Jr (2003) Shikimate accumulates in both glyphosate-sensitive and glyphosate-resistant horseweed (Conyza canadensis L. Cronq.). J Agric Food Chem 51:680–684
Mueller TC, Ellis AT, Beeler JE, Sharma SD, Singh M (2008) Shikimate accumulation in nine weedy species following glyphosate application. Weed Res 48:455–460
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassarys with tobacco tissue cultures. Physiol Plant 15:473–497
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279
Papanikou E, Brotherton JE, Widholm JM (2004) Length of time in tissue culture can affect the selected glyphosate resistance mechanism. Planta 218:589–598
Perez-Jones A, Park KW, Polge N, Colquhoun J, Mallory-Smith CA (2007) Investigating the mechanisms of glyphosate resistance in Lolium multiflorum. Planta 226:395–404
Pline-Srnic W (2006) Physiological mechanisms of glyphosate resistance. Weed Technol 20:290–300
Pratley J, Urwin N, Stanton R, Bainers P, Broster J, Cullis K, Schafer D, Bohn J, Krueger R (1999) Resistance to glyphosate in Lolium rigidum. I. Bioevaluation. Weed Sci 47:405–411
Sergiev IG, Alexieva VS, Ivanov SV, Moskova II, Karanov EN (2006) The phenylurea cytokinin 4PU-30 protects maize plants against glyphosate action. Pest Biochem Phys 85(3):139–146
Shaner DL, Nadler-Hassar T, Henry WB, Koger CH (2005) A rapid in vivo shikimate accumulation assay with excised leaf discs. Weed Sci 53(6):769–774
Simarmata M, Kaufmann JE, Penner D (2003) Potential basis of glyphosate resistance in California rigid ryegrass (Lolium rigidum). Weed Sci 51:678–682
Smirnoff N (1993) The role of active oxygen in the responses of plants to water deficit and desiccation. New Phytol 125:27–58
Steinrucken HC, Amrhein N (1980) The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochem Biophys Res Commun 94:1207–1212
Subramanyam K, Sailaja KV, Subramanyam K, Rao DM, Lakshmidevi K (2011) Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annum L.). Plant Cell Tiss Organ Cult 105:181–192
Sun J, Li LS, Liu MQ, Wang MJ, Ding MQ, Deng SR, Lu CF, Zhou XY, Shen X, Zheng XJ, Chen SL (2010) Hydrogen peroxide and nitric oxide mediate K+/Na+ homeostasis and antioxidant defence in NaCl-stressed callus cells of two contrasting poplars. Plant Cell Tiss Organ Cult 103:205–215
Tait MA, Hik DS (2003) Is dimethylsulfoxide a reliable solvent for extracting chlorophyll under field conditions? Photosynth Res 78:87–91
Tran M, Baerson S, Brinker R, Casagrande L, Faletti M, Feng Y, Nemeth M, Reynolds T, Rodriguez D, Shaffer D, Stalker D, Taylor N, Teng Y, Dill G (1999) Characterization of glyphosate resistant Eleusine indica biotypes from Malaysia. In: Proceedings 1 (B) of the 17th Asian-Pacific weed science society conference. The Asian-Pacific Weed Science Society, Bangkok, pp 527–536
Wakelin AM, Preston C (2006) A target-site mutation is present in a glyphosate-resistant Lolium rigidum population. Weed Res 46:432–440
Yanniccari M, Istilart C, Giménez DO, Castro AM (2012) Glyphosate resistance in perennial ryegrass (Lolium perenne L.) from Argentina. Crop Prot 32:12–16
Zapiola ML, Campbell CK, Butler MD, Mallory-Smith CA (2008) Escape and establishment of transgenic glyphosate-resistant creeping bentgrass Agrostis stolonifera in Oregan, USA: a 4-year study. J App Ecol 45:486–494
Zhang GY, Lu S, Chen TA, Funk CR, Meyer W (2003) Transformation of triploid bermudagrass (Cynodon dactylon × C. transvaalensis cv. TifEagle) by means of biolistic bombardment. Plant Cell Rep 21:860–864
Zhang SG, Han SY, Yang WH, Wei HL, Zhang M, Qi LW (2010) Changes in H2O2 content and antioxidant enzyme gene expression during the somatic embryogenesis of Larix leptolepis. Plant Cell Tiss Organ Cult 100:21–29
Acknowledgments
This study was carried out with financial support from the National Science Foundation of China (30771522). Andrej Svyantek is acknowledged for revising the English in the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chen, S., Chai, M., Jia, Y. et al. In vitro selection of glyphosate-tolerant variants from long-term callus cultures of Zoysia matrella [L.] Merr. Plant Cell Tiss Organ Cult 111, 199–207 (2012). https://doi.org/10.1007/s11240-012-0184-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-012-0184-z