Effect of inoculation times and acetosyringone concentrations on gene transfer efficiency of β-glucuronidase (GUS) gene mediated by Agrobacterium in gerbera (Gerbera jamesonii cv. ‘Royal Soft Pink’)

Document Type : original paper

Abstract

Background and Objectives: Gerbera is one of the most popular ornamental plants in the world and ranked forth, in the flower industry among the 10 top cut flowers. Nowadays, there is an important for development of generally applicable gene transfer methods for gerbera which will allow rapid introduction of new traits into elite genotypes without changing the existing good properties.
Materials and methods: This study was conducted in three separate experiments; First experiment was conducted to evaluate the effect of hgygromaycin at various concentrations (5, 10, 15, 20, 25 and 30 mg L-1) to determine it’s the lethal dose in untransformed plantlets of Gerbera jamesonii cv. ‘Royal Soft Pink’. Then, in second experiment investigated the effect of 10, 15, 20, 25 and 30 mg L-1 hgygromaycin on shoot regeneration in leafy petiole explants. Finally, in third experiment evaluated the effect of inoculation times (10 and 20 min) and acetosyringone concentrations (50 and 100 µmol) on GUS gene transfer efficiency mediated by Agrobacterium and by co-cultivation method of leafy petiole explants. After 2-3 days of co-cultivation of leafy petiole explants with Agrobacterium, were transferred to direct shoot regeneration medium. Then putative transgenic shoots derived from petioles and the multiplication were done. Finally, transformed plants were confirmed by GUS histochemical assay and PCR analysis.
Results: The results showed that the 10 mg L-1 concentration of hgygromaycin is suitable for selection of transgenic shoots and it should be used after initial regeneration. The highest (38%) of putative transgenic shoots regeneration was obtained from treatment of 10 min inoculation with Agrobacterium and using 100 µmol acetosyringone. Also, the lowest (25.25%) of putative transgenic shoots regeneration was showed from treatments of 20 min inoculation and using 50 µmol acetosyringone. The results of histochemical assay and PCR analysis in transgenic plants showed that the highest independent line number (27 shoots) and gene transfer efficiency (11%) per 100 explants were obtained from treatments of 10 min inoculation and using 100 µmol acetosyringone.
Conclusion: Factors such as inoculation time and acetosyringone concentration had prominent impact on the efficiency and success of transformation. Acetosyringone, is considered to be the phenolic inducer of vir genes activation and T-DNA transfer in Agrobacterium and enhances the transgenic efficiency. Therefore, the best treatment to produce transgenic plant in Gerbera jamesonii cv. ‘Royal Soft Pink’ with transfer of GUS gene is the inoculation of leafy petiole explants for 10 min and using of acetosyringone with 100 µmol concentration.

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References

1.Ahmadpour, R., Zare, N., Asghari-Zakaria, R. and Sheikhzadeh, P. 2015. Enhancement of
Agrobacterium-mediated transformation efficiency in immature embryo of Triticum
aestivum cv. Arya. Irn. J. Genet. Plant Breed. 4: 45-53.
2.Almeida, W.A.B., Filho, F.A.A.M., Mendes, B.M.J., Pavan, A. and Rodriguez, A.P.M. 2003.
Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl
segments. Sci. Agri. 60: 23-29.
3.Amoah, B.K., Wu, H., Sparks, C. and Jones, H.D. 2001. Factors influencing Agrobacteriummediated
transient expression of uidA in wheat inflorescence tissue. J. Exp. Bot. 52: 1135-1142.
4.Aswath, C.R., Mo, S.Y., Kim, S.H. and Kim, D.H. 2004. IbMADS4 regulates the vegetative
shoot development in transgenic chrysanthemum (Dendranthema grandiflora (Ramat.)
Kitamura). Plant Sci. 166: 847-854.
5.Azadi, P., Bagheri, H., Nalousi, A.M., Nazari, F. and Chandler, S.F. 2016. Current status
and biotechnological advances in genetic engineering of ornamental plants. Biotechnol. Adv.
34: 1073-1090.
6.Berendzen, K., Searle, I., Ravenscroft, D., Koncz, C., Batschauer, A., Coupland, G.,
Somssich, I.E. and Ülker, B. 2005. A rapid and versatile combined DNA/RNA extraction
protocol and its application to the analysis of a novel DNA marker set polymorphic between
Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta. Plant Meth.4: 1-15.
7.Chakravarty, B. and Wang-Pruski, G. 2010. Rapid regeneration of stable transformants in
cultures of potato by improving factors influencing Agrobacterium-mediated transformation.
Adv. Biosci. Biotechnol. 1: 409-416.
8.Costa, M.G.C., Otoni, W.C. and Moore, G.A. 2002. An evaluation of factors affecting the
efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and
production of transgenic plants containing carotenoid biosynthetic genes. Plant Cell Rep.
21: 365-373.
9.Dai, Y.M., Lin, J.B., Wang, W.Y., Zou, H., Wu, S.J. and Lin, Y.X. 2010. Transformation of
blue gene in Narcissus tazetta var. chinensis mediated by Agrobacterium tumefaciens.
J. Agri. Biotechnol. 18: 231-238.
10.Dewir, Y.H., El-Mahrouk, M.E. and El-Banna, A.N. 2015. In vitro propagation and
preliminary results of Agrobacterium-mediated genetic transformation of Cordyline
fruticosa. S. Afr. J. Bot. 98: 45-51.
11.Dutt, M. and Grosser, J.W. 2009. Evaluation of parameters affecting Agrobacteriummediated
transformation of citrus. Plant Cell Tiss. Org. Cult. 98: 331-340.
12.Elomaa, P. and Teeri, T.H. 2001. Transgenic gerbera. In: Biotechnol. Agri. Forest.
48: Transgenic Crops III (Bajaj, Y.P.S., Ed.), Springer-Verlag, Berlin. Pp: 139-154.
13.Godwin, I., Todd, G., Ford-Lloyd, B. and Newbury, H.J. 1991. The effects of acetosyringone
and pH on Agrobacterium mediated transformation vary according to plant species. Plant
Cell Rep. 9: 671-675.
14.Gonzalez, A., Jimenez, A., Vazquez, D., Davies, J. and Schindles, D. 1987. Studies on the
mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochim.
Biophys. Acta – Nucleic Acids Protein Synth. 521: 459-469.
15.Gutlitz, R.G.H., Lamb, P.W. and Matthsse, A.G. 1987. Involvement of carrot cell surface
proteins in attachment of Agrobacterium tumefaciens. Plant Physiol. 83: 564.
16.Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusions: β-glucoronidase as a
sensitive and versatile gene fusion marker in higher plants. The EMBO J. 6: 3901-3907.
17.Kalawong, S., Srichuay, W. and Te-chato, S. 2014. The effect of Agrobacterium densities
and inoculation times on gene transformation efficiency in rubber tree. Afr. J. Biotechnol.
13: 2321-2329.
18.Kondo, T., Hasegawa, H. and Suzuki, M. 2000. Transformation and regeneration of garlic
(Allium sativum L.) by Agrobacterium-mediated gene transfer. Plant Cell Rpt. 19: 989-993.
19.Koushesh Saba, M. and Nazari, F. 2017. Vase life of gerbera cut flower cv. Pink Power
affected by different treatments of plant essential oils and silver nanoparticles. J. Plant Prod.
Res. 2: 43-59.
20.Mathews, H., Bhararhan, N., Litz, R.E., Narayanan, P.S. and Bharia, C.R. 1990. The
promotion of Agrobacterium-mediated transformation in Atropa belladona L. by
acetosyringone. J. Plant Physiol. 136: 404-409.
21.Miki, B. and McHugh, S. 2004. Selectable marker genes in transgenic plants: applications,
alternatives and biosafety. J. Biotechnol. 107: 193-232.
22.Nazari, F., Khosh-Khui, M. and Azadi, P. 2016. A simple and efficient direct shoot
organogenesis method using leafy petiole explants in Gerbera jamesonii cv. Royal Soft Pink.
Int. J. Hort. Sci. Technol. 3: 51-58.
23.Nazari, F., Khosh-Khui, M. and Azadi, P. 2017. Production of delphinidin anthocyanin in
petals of gerbera flower by agroinfiltration of flower color gene constructs. J. Plant Prod.
Res. 23: 145-164.
24.Nazari, F., Khosh-Khui, M., Azadi, P., Salehi, H. and Niazi, A. 2014. Growth regulators
affected in vitro propagation of pot gerbera (Gerbera jamesonii cv. Royal Soft Pink). Int. J.
Agri. Biosci. 3: 185-189.
25.Neskorodov, Y.B., Rakitin, A.L., Kamionskaya, A.M. and Skryabin, K.G. 2010) Developing
phosphinothricin-resistant transgenic sunflower (Helianthus annuus L.) plants. Plant Cell
Tiss. Organ Cult. 100: 65-71.
26.Ohta, S., Mita, S., Hattori, T. and Nakamura, K. 1990. Construction and expression in
tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding
sequences. Plant Cell Physiol. 31: 805-813.
27.Pandey, V., Misra, P., Chaturvedi, P., Misra, M.K., Trivedi, P.K. and Tull, R. 2010.
Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: An
important medicinal plant. Plant Cell Rpt. 29: 133-141.
28.Park, S.H., Perison, S.R.M. and Smith, R.H. 1996. T- DNA integration into genomic DNA of
rice following Agrobacterium inoculation of isolated shoot apices. Plant Mol. Biol.
32: 1135-1148.
29.Parveez, G.K.A., Majid, N.A., Zainal, A. and Rasid, O.A. 2007. Determination of minimal
inhibitory concentration of selection agents for selecting transformed immature embryos of
oil palm. AsPac J. Mol. Biol. Biotechnol. 15: 133-146.
30.Ramzan Khan, M., Rashid, H., Ansar, M. and Chaudry, Z. 2003. High frequency shoot
regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus). Plant
Cell Tiss. Organ Cult. 75: 223.
31.Sánchez-Velázquez, J.U., Puc, G.L., Ramos-Díaz, A.L., Cano-Sosa, J.S., Buenfil, I.M.R.,
García-Velasco, R. and Varguez, A.U. 2016. Main factors affecting the genetic
transformation of chrysanthemum var. Micromargara. Plant Omics J. 9: 121-125.
32.Sawahel, W. and Hagran, A. 2006. Generation of white mold disease resistant sunflower
plants expressing human lysozyme gene. Biol. Plant. 50: 683-687.
33.Sheikholeslam, S.N. and Weeks, D.P. 1987. Acetosyringone promotes high efficiency
transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol.
Biol. 8: 291-298.
34.Shen, W.H., Escudero, J., Schlappi, M., Ramos, C., Hoha, B. and Koukolikova-Nicola, Z.
1993. TDNA transfer to maize cells: histochemical invbestigation of β-glucuronidase activity
in maize tissues. Proc. Natl. Acad. Sci. U.S.A. 90: 1488-1492.
35.Sujatha, M., Vijay, S., Vasavi, S., Reddy, P.V. and Rao, S.C. 2012. Agrobacterium-mediated
transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus
annuus L.). Plant Cell Tiss. Organ Cult. 110: 275-287.
36.Sun, C.Y., Wang, Y., Xu, X.F., Sun, Y., Zhu, L.H. and Han, Z.H. 2008. Regeneration
from leaf segments of in vitro-grown shoots of Malus baccata. New Zeal. J. Crop Hort. Sci.
36: 233-238.
37.Sundar, I.K. and Sakthivel, N. 2008. Advances in selectable marker genes for plant
transformation. J. Plant Physiol. 165: 1698-1716.
38.Sunilkumar, G. and Rathore, K.S. 2001. Transgenic cotton: factors influencing
Agrobacterium mediated transformation and regeneration. Mol. Breed. 8: 37-52.
39.Sunilkumar, G., Vijaychandra, K. and Veluthambi, K. 1999. Pre-incubation of tobacco leaf
explant promotes Agrobacterium mediated transformation by increasing vir gene induction.
Plant Sci. 141: 51-58.
40.Yang, Z., Ingrelbrecht, N., Louzada, I.L., Skaria, E. and Mirkov, T.E. 2000. Agrobacteriummediated
transformation of the commercially important grapefruit cultivar Rio Red
(Citrus paradisi Macf.). Plant Cell Rep. 11: 1203-1211.
41.Zhu, Y.L. and Ma, F.W. 2009. Establishment of Agrobacterium-mediated transformation
system for Cyclamen persicum. Acta Agri. Boreali-occidentalis Sinica. 18: 240-244.
Journal of Plant Production Research
Volume 25, Issue 2
September 2018
Pages 53-66

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