Genetic transformation of Persian melon (Cucumis melo L.) via Agrobacterium

Document Type : Research paper

Authors

1 Department of Horticulture, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran.

2 Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

3 Department of Horticulture, College of Aburaihan, University of Tehran, Pakdasht, Tehran, Iran.

Abstract

A reliable Agrobacterium-mediated transformation and regeneration protocol was developed for commercially important endemic Persian melon cultigens (Cucumis melo L.) comprising ‘Eyvanaki’, ‘Samsoori’, and ‘Khatooni’. The effect of selective Murashige and Skoog (MS) medium containing various concentrations of 6-benzyl adenine (BA) (0, 0.5, 1, and 1.5 mg l-1) and 1 mg l-1 Gibberellic acid (GA3) on regeneration of cotyledon, hypocotyl, and cotyledonary petioles derived from 6-day-old in vitro grown seedlings of the three Persian melons were investigated. For transformation, the sensitivity to kanamycin (Km) concentrations (0, 50, 75, 100, 125 mg l-1 ), the effect of three A. tumefaciens strains (GV3103, LBA4404, and AGL0), inoculation time (0.5, 1, 5, and 30 min), and co-cultivation time (24, 48, and 72 h) on direct shoot regeneration of cotyledonary petiole of ‘Samsoori’ were investigated. Shoot regeneration from cotyledonary petiole explants received the highest attention. Cotyledonary petiole segments of ‘Samsoori’ and ‘Khatooni’ treated respectively with 1.0 mg l-1 and 1.5 mg l-1 BA exhibited the highest potential for shoot multiplication; while the regeneration rate of ‘Eyvanaki’ was drastically lower. Putative transgenic ‘Samsoori’ plantlets selected in 100 mg l-1 Km were subcultured on elongation MS medium composed of 100 mg l-1 Km, 0.1 mg l-1 BA, 1 mg l-1 GA3 plus 400 mg l-1 CTX, and then successfully rooted on growth regulator-free MS medium for two weeks. Using histochemical GUS assay along with genomic PCR screening for GusA and VirG genes, the efficiency of transformation was estimated to be 10% for AGL0 and 6% in LBA4404.

Keywords


Adiguzel P., Nyirahabimana F., Shimira F, Solmaz I., and Taskin H. (2023). Applied biotechnological approaches for reducing yield gap in melon grown under saline and drought stresses: an overview. Journal of Soil Science and Plant Nutrition, 23(1): 139-51.
Agrawal S., and Rami E. (2022). A Review: Agrobacterium-mediated gene transformation to increase plant productivity. The Journal of Phytopharmacology, 11(2): 111-117. DOI: 10.31254/phyto.2022.11211.
Akasaka-Kennedy Y., Tomita K.-O., and Ezura H. (2004). Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in melon (Cucumis melo L.). Plant Science, 166(3): 763-769.
Bezirganoglu I., Hwang S. Y., Shaw J. F., and Fang T. J. (2014). Efficient production of transgenic melon via Agrobacterium-mediated transformation. Genetics and Molecular Research, 13(2): 3218-27. 
Boszoradova E., Libantova J., Matusikova I., Poloniova Z., Jopcik M., Berenyi M., and Moravcikova J. (2011). Agrobacterium-mediated genetic transformation of economically important oilseed rape cultivars. Plant Cell, Tissue and Organ Culture, 107: 317-323.
Box G. E., Hunter J. S., and Hunter W. G. (2005). Statistics for experimenters. In: Wiley Series in Probability and Statistics Hoboken, NJ: Wiley.
Burger Y., Saar U., Distelfeld A., Katzir N., Yeselson Y., Shen S., and Schaffer A. A. (2003). Development of sweet melon (Cucumis melo) genotypes combining high sucrose and organic acid content. Journal of the American Society for Horticultural Science, 128: 537-540.
Choi J. Y., Shin J. S., Chung Y. S., and Hyung N. I. (2012). An efficient selection and regeneration protocol for Agrobacterium-mediated transformation of oriental melon (Cucumis melo L. var. makuwa). Plant Cell Tissue & Organ Culture, 110: 133-140. DOI: 10.1007/s11240-012-0137-6. 
Choudhary H., Yadav R. K., and Maurya S. K. (2020). Principles and Techniques for rapid improvement of muskmelon for yield, fruit quality and resistance to biotic stresses. In book: Accelerated Plant Breeding, Volume 2, Vegetable Crops, 373-395.
Chovelon V., Restier V., Dogimont C., Aarouf J., and Pitrat M. (2008). Histological study of shoot organogenesis in melon (Cucumis melo L.) after genetic transformation. Pitrat M. (Ed.): Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae, Avignon (France), May 21-24th, 633-637.
Curuk S., Cetiner S., Elman C., Xia X., Wang Y., Yeheskel A., Zilberstein L., Perl‐Treves R., Watad A., and Gaba V. (2005). Transformation of recalcitrant melon (Cucumis melo L.) cultivars is facilitated by wounding with carborundum. Engineering in Life Sciences, 5: 169-177. DOI: 10.1002/elsc.200520069. 
Curuk S., Elman C., Schlarman E., Sagee O., Shomer I., Cetiner S., Gray D., and Gaba V. (2002). A novel pathway for rapid shoot regeneration from the proximal zone of the inpocotyl of melon (Cucumis melo L.). In Vitro Cellular & Developmental Biology – Plant, 38: 260-267. DOI: 10.1079/ivp2001259. 
Danesh M., Lotfi M., and Azizinia S. (2015). Genetic diversity of Iranian melon cultigens revealed by AFLP markers. International Journal of Horticulture Science & Technology, 2: 43-53.
De la Rosa R., James C. M., and Tobutt K. R. (2002). Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae. Molecular Ecology Notes, 2(3): 265-267. DOI: https://doi.org/10.1046/j.1471- 8286.2002.00217.x.
Dobson J. (2006). Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery. Gene Therapy, 13(4): 283-287.
Dogimont C. (2011). Gene list for melon. Cucurbit Genetics Cooperative Report, 33: 104-133.
Feng J., Wang N., Li Y., Wang H., Zhang W., Wang H., and Chai S. (2023). Recent progress in genetic transformation and gene editing technology in cucurbit crops. Agronomy, 13(3): 755.
Garcia-Almodovar R. C., Gosalvez B., Aranda M. A., and Burgos L. (2017). Production of transgenic diploid Cucumis melo plants. Plant Cell Tissue & Organ Culture, 130: 323-333. DOI: https://doi.org/10.1007/s11240-017-1227-2.
Grozeva S. Y., Velkov N. V., and Ivanova Z. V. (2019). In vitro plant regeneration of two Cucumis melo L. genotypes using different explant types and culture medium. Ecologia Balkanica, 11(2): 193-202.
Guis M., Amor M. B., Latché A., Pech J. C., and Roustan J. P. (2000). A reliable system for the transformation of cantaloupe charentais melon (Cucumis melo L. var. cantalupensis) leading to a majority of diploid regenerants. Scientia Horticulturae, 84: 91-99. DOI: 10.1016/s0304-4238(99)00101-6. 
Hoekema A., Hirsch P., Hooykaas P., and Schilperoort R. (1983). A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 303: 179-180. DOI: 10.1038/303179a0. 
Jefferson R. A., Kavanagh T. A., and Bevan M. W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal, 6: 3901-3907.
Kesh H., and Kaushik P. (2021). Advances in melon (Cucumis melo L.) breeding: An update. Scientia Horticulturae, 282: 110045.
Komala M. A., and Kuni P. (2022). Genetic diversity and molecular breeding of melon (Cucumis melo L.): A Review. Current Agriculture Research Journal, 10(3): 181-92.
Lazo G. R., Stein P. A., and Ludwig R. A. (1991). A DNA transformation–competent Arabidopsis genomic library in Agrobacterium. Nature Biotechnology, 9: 963-967.
Li D., Wei X., Liu T., Liu C., Chen W., Xuan Y. H., and Gao L. (2020). Establishment of an Agrobacterium tumefaciens-mediated transformation system for Tilletia foetida. Journal of Microbiological Methods, 169: 105810.
Lotfi M., and Kashi A. (1999). The Iranian melon as a new cultivar group. In: Taxonomy of Cultivated Plants, Third International Symposium, Royal Botanic Gardens, Kew, 447-449.
Mallek‐Ayadi S., Bahloul N., Baklouti S., and Kechaou N. (2022). Bioactive compounds from Cucumis melo L. fruits as potential nutraceutical food ingredients and juice processing using membrane technology. Food Science & Nutrition, 10(9): 2922-34. 
Mousavi M., Mousavi A., Habashi A. A., and Dehsara B. (2014). Genetic transformation of date palm (Phoenix dactylifera L. cv. ‘Estamaran’) via particle bombardment. Molecular Biology Reports, 41: 8185-8194. DOI: 10.1007/s11033-014-3720-6. 
Murashige T., and Skoog F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Plant Physiology, 15: 473-497. DOI: 10.1111/j.1399-3054.1962.tb08052.x. 
Navratilova B., Skalova D., Ondrej V., Kitner M., and Lebeda A. (2011). Biotechnological methods utilized in Cucumis research-A review. Horticultural Science, 38(4): 150-8.
Nonaka S., Yuhashi K. I., Takada K., Sugaware M., Minamisawa K., and Ezura H. (2008). Ethylene production in plants during transformation suppresses vir gene expression in Agrobacterium tumefaciens. New Phytologist, 178: 647-656. DOI: 10.1111/j.1469-8137.2008.02400.x. 
Nora F. R., Peters J. A., Schuch M. W., Lucchetta L., Marini L., Jorge A., and Rombaldi C. V. (2001). Melon regeneration and transformation using an apple ACC oxidase antisense gene. Current Agricultural Science and Technology, 7(3): 201-204.
Nuñez-Palenius H. G., Gomez-Lim M., Ochoa-Alejo N., Grumet R., Lester G., and Cantliffe D. J. (2008). Melon fruits: genetic diversity, physiology, and biotechnology features. Critical Reviews in Biotechnology, 28: 13-55. DOI: 10.1080/07388550801891111. 
Pushyami B., Beena M. R., Sinha M. K., and Kirti P. B. (2011). In vitro regeneration and optimization of conditions for Agrobacterium-mediated transformation in jute, Corchorus capsularisJournal of Plant Biochemistry and Biotechnology, 20: 39-46.
Radchuk V., Klocke E., Radchuk R., Neumann M., and Blume Y. (2000). Production of transgenic rape plants (Brassica napus L.) using Agrobacterium tumefaciens. Genetika, 36: 932-941.
Raji M. R., Lotfi M., Tohidfar M., Ramshini H., Sahebani N., Aalifar M., Baratian M., Mercati F., De Michele R., and Carimi F. (2022). Multiple fungal diseases resistance induction in Cucumis melo through co-transformation of different pathogenesis-related (PR) protein genes. Scientia Horticulturae, 297: 110924.
Robinson R., and Decker-Walters D. (1999). Cucurbits. CAB International, Wallingford. Oxford, UK.
Royston P. (1992). Approximating the Shapiro-Wilk W-Test for non-normality. Statistics & Computing, 2: 117-119. DOI: 10.1007/bf01891203. 
Selvaraj N., Kasthurirengan S., Vasudevan A., Manickavasagam M., Choi C., and Ganapathi A. (2010). Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (Cucumis sativus L. cv. Poinsett76) via Agrobacterium tumefaciens. In Vitro Cellular & Developmental Biology – Plant, 46: 329-337. DOI: 10.1007/s11627-010-9288-5. 
Shirazi Parsa H., Sabet M. S., Moieni A., Shojaeiyan A., Dogimont C., Boualem A., and Bendahmane A. (2023). CRISPR/Cas9-Mediated Cytosine base editing using an improved transformation procedure in melon (Cucumis melo L.). International Journal of Molecular Sciences, 24(13): 11189.
Silva J. D., and Fukai S. (2001). The impact of carbenicillin, cefotaxime and vancomycin on chrysanthemum and tobacco TCL morphogenesis and Agrobacterium growth. Journal of Applied Horticulture, 3: 3-12.
Silva M. A., Albuquerque T. G., Alves R. C., Oliveira M. B., and Costa H. S. (2020). Melon (Cucumis melo L.) by-products: Potential food ingredients for novel functional foods? Trends in Food Science & Technology, 98: 181-9.
Steel R. G., and Dickey J. H. (1997). Principles and procedures of statistics: A biometrical approach. WCB/McGraw-Hill, Boston, Massachusetts, USA, pp. 672.
Sutradhar M., and Mandal N. (2023). Reasons and riddance of Agrobacterium tumefaciens overgrowth in plant transformation. Transgenic Research, 32(1-2): 33-52.
Takavar S., Rahnama H., Rahimian H., and Kazemitabar K. (2010). Agrobacterium-mediated transformation of maize (Zea mays L.). Journal of Sciences Islamic Republic of Iran, 21: 21-29.
Valles M., and Lasa J. (1994). Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro). Plant Cell Reports, 13: 145-148. DOI: 10.1007/bf00239881. 
Vengadesan G., Anand R. P., Selvaraj N., Perl-Treves R., and Ganapathi A. (2005). Transfer and expression of nptII and bar genes in cucumber (Cucumis satavus L.). In Vitro Cellular & Developmental Biology – Plant, 41: 17-21. DOI: 10.1079/ivp2004602.
Wan L., Wang Z., Zhang X., Zeng H., Ren J., Zhang N., Sun Y., and Mi T. (2023). Optimized Agrobacterium-mediated transformation and application of developmental regulators improve regeneration efficiency in melons. Genes, 14(7): 1432. 
Wang S. L., Ku S. S., Ye X. G., He C. F., Kwon S. Y., and Choi P. S. (2015). Current status of genetic transformation technology developed in cucumber (Cucumis sativus L.). Journal of Integrative Agriculture, 14: 469-482. DOI: 10.1016/s2095-3119(14)60899-6. 
Wiebke B., Ferreira F., Pasquali G., Bodanese-Zanettini M. H., and Droste A. (2006). Influence of antibiotics on embryogenic tissue and Agrobacterium tumefaciens suppression in soybean genetic transformation, Bragantia, 65: 543-551. DOI: 10.1590/s0006-87052006000400002. 
Xiao L., Zhao Q., Cao X., Yao Z., and Zhao S. (2023). The TIR-Type NLR protein is involved in the regulation of Phelipanche aegyptiaca resistance in Cucumis meloAgronomy, 13(3): 644.
Zhang H., Gao P., Wang X., and Luan F. (2014). An improved method of Agrobacterium tumefaciens-mediated genetic transformation system of melon (Cucumis melo L.). Journal of Plant Biochemistry & Biotechnology, 23: 278-283. DOI: 10.1007/s13562-013-0211-0.