TURUNÇGİL ISLAHI: EMBRİYO KÜLTÜRÜ ÇALIŞMALARI

Öz

Çalışmada, turunçgil ıslahında kullanılan geleneksel yöntemlerden seleksiyon, melezleme ve mutasyon teknikleri ile birlikte veya tek başına kullanılarak daha kısa zamanda ve daha hızlı ilerleme sağlayan biyoteknolojik yöntemlerden, ploidi ıslahı, izoenzim analizleri, moleküler teknikler, genetik transformasyon, somatik melezleme ve özellikle embriyo kültürü ile bu tekniğe etki eden faktörler irdelenmiştir. Geleneksel turunçgil ıslahı; uyuşmazlık, heterozigoti, apomiksis, poliembriyoni ve uzun süren gençlik kısırlığına bağlı olarak zahmetli, maliyetli ve zaman alıcı bir uğraştır. Islah çalışmalarında ister geleneksel ister modern teknikler olsun hedefe ulaşmak için uygun ebeveynlerin ve uygun tekniklerin seçimi önemlidir. Ebeveynler arasındaki genetik ilişkiler ile hedeflenen karakterlerin kalıtımı hakkında elde edilen bilgiler, ıslah çalışmalarının doğru yönlendirilmesi ve daha kısa sürede başarı sağlanması için oldukça önemlidir. Poliembriyoni nedeniyle yeni melez bireylerin oluşumu sınırlı olmasına rağmen melezleme ıslahı, turunçgillerde çeşit geliştirmenin temelini oluşturmaktadır. Poliembriyonik turunçgillerde zigotik embriyo, nuseller embriyolarla besin maddesi ve gelişim alanı için yarışmak zorundadır ve nuseller embriyolar, zigotik embriyoların gelişimini baskılamaktadır. Embriyo kurtarma tekniği, kontrollü melezlemeler ve somatik melezlemelerde bu sorunun çözümü için gerekli ve yararlı bir metottur. Bu tekniğin başarısını; embriyo gelişim aşaması, kültür ortamının bileşimi, embriyo alım tekniği ve genotip etkilemektedir. In vitro yöntemler ve moleküler tekniklerdeki yeni gelişmeler ve bunların ıslah programlarında kullanımının yaygınlaşması turunçgillerde yeni çeşitlerin elde edilmesine olanak sağlayacaktır.

Anahtar Kelimeler

• citrus
• ıslah
• biyoteknoloji
• doku kültürü
• embriyo kurtarma

CITRUS BREEDING: EMBRYO CULTURE STUDIES

Abstract

In this study, it was evaluated important factors for conventional breeding techniques (selection, hybridization artificial mutation) and biotechnological methods (ploidy breeding, isoenzyme analysis, molecular techniques, genetic transformation, somatic hybridization and especially embryo culture) in citrus breeding. Conventional citrus breeding is laborious, costly and time-consuming due to incompatibility, heterozygous, apomixis, polyembryony and prolonged juvenility. It is important to choose suitable parents and suitable techniques to achieve the goal in breeding studies whether traditional or modern techniques. Obtained knowledge about the genetic relationships between parents and the inheritance of the targeted characters is important for the manipulated of breeding studies and achievement in a shorter time. Hybridization breeding is the basis of developing cultivars in citrus, although the obtained of new hybrid genotypes is limited because of polyembryony. The zygotic embryo must compete with the nuceller embryos for nutrient and growth area in polyembryonic citrus cultivars and the nuceller embryos suppress the development of the zygotic embryos. Embryo rescue technique is a necessary and useful method for solving this problem in controlled hybridization and somatic hybridization. The success of this technique is affected by the embryo development stage, composition of the culture medium, embryo excise technique and genotype. New developments in in vitro methods and molecular techniques and the widespread use of these in breeding programs will provide an opportunity breeding new cultivars in Citrus.

Keywords

• citrus
• breeding
• biotechnology
• tissue culture
• embryo rescue

Kaynakça

1. Ahmed, S., H.S. Rattanpal, P. Kumari and J. Singh, 2017. Study of genetic variability in Citrus fruit crop by molecular markers. International Journal of Pure and Applied Bioscience, 5(1):111-128.
2. Aka-Kaçar, Y., A. Uzun, İ. Polat, T. Yeşiloğlu, B. Yılmaz, O. Gülşen, Ö. Tuzcu, M. Kamiloğlu, Ş. Kurt and U. Seday, 2013. Molecular characterization and genetic diversity analysis of mandarin genotypes by SSR and SRAP markers. Journal of Food, Agriculture and Environment, 11(1):516-521.
3. Aleza, P., J. Juárez, J. Cuenca, P. Ollitrault and L. Navarro, 2010a. Recovery of Citrus triploid hybrids by embryo rescue and flow cytometry from 2x × 2x sexual hybridization and its application to extensive breeding programs. Plant Cell Reports, 29(9):1023-1034.
4. Aleza, P., J. Juárez, P. Ollitrault and L. Navarro, 2010b. Polyembryony in non-apomictic Citrus genotypes. Annals of Botany, 106(4):533-545.
5. Aleza, P., J. Juárez, J. Cuenca, P. Ollitrault and L. Navarro, 2012. Extensive Citrus triploid hybrid production by 2x × 4x sexual hybridizations and parent-effect on the length of the juvenile phase. Plant Cell Reports, 31:1723-1735.
6. Allario, T., J. Brumos, J.M. Colmenero-Flores, D.J. Iglesias, J.A. Pina, L. Navarro, M. Talon, P. Ollitrault and R. Morillon, 2012. Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production. Plant, Cell and Environment, 36(4):856-68.
7. Anonim, 2019a. 2017 yılı turunçgil üretim verileri. Food and Agriculture Organization (FAO). (http://www.fao.org/ faostat; Erişim Tarihi: Mart 2020).
8. Anonim, 2019b. 2018 yılı Türkiye turunçgil üretim verileri. Türkiye İstatistik Kurumu (TÜİK) (https://biruni.tuik.gov.tr/ bitkiselapp/bitkisel.zul; Erişim Tarihi: Mart 2020).

9. Anonim, 2019c. 2019 yılı tescilli meyve ve asma çeşit listesi. Tohumluk Tescil ve Sertifikasyon Merkez Müdürlüğü (https:// www.tarimorman.gov.tr/bugem/ttsm; Erişim Tarihi: Mart 2020).
10. Bhojwani, S.S. and M.K. Razdan, 1983. Plant tissue culture: theory and practice. Elsevier, 502p.
11. Bijzet, Z., 2006. Pollination. In: E.A. De Villiers (Ed.): The Cultivation of Citrus. ARC-Institute for Tropical and Subtropical Crops, pp:105-122.
12. Bridgen, M.P., 1994. A review of plant embryo culture. Hort.Science, 29(11): 1243-1246.
13. Buffard-Morel, J., 1968. Effets du glucose, du lévulose, du maltose et du saccharose sur le développement des embryons de palmier à huile (Elaeis guineensis Jacq. var. Dura Bec.) en culture in vitro. Comptes Rendus de l’Académie des Sciences, 267D:185-188.
14. Cameron, J.W. and H.B. Frost, 1968. Genetics, breeding and nucellar embryony. In: W. Reuther, L.D. Batchelor, H.J. Webber (Eds.): The Citrus Industry. Vol. II. Anatomy, Physiology, Genetics, and Reproduction. University of California, Division of Agricultural Sciences, pp:325-370.
15. Cardoso, J.C., A.M. Abdelgalel, B. Chiancone, R.R. Latadoc, O. Laine, R. Testoline and M.A. Germana, 2015. Gametic and somatic embryogenesis through in vitro anther culture of different Citrus genotypes. Biosystems-An International Journal Dealing with all Aspects of Plant Biology, 150(2):304-312.
16. Carimi, F., F. Pasquale and A.M. Puglia, 1998. In vitro rescue of zygotic embryos of sour orange (Citrus aurantium L.) and their detection based on RFLP analysis. Plant Breeding, 117:261-266.
17. Castle, W.S., 2010. A career perspective on Citrus rootstocks, their development and commercialization. Hort. Science, 45: 11-15.
18. Chagas, E.A., M. Pasqual, J.D. Ramos, L.A.S. Pio, L.F. Dutra and J.O. Cazetta, 2005. Activated charcoal and gibberellic acid concentrations on immature embryos culture. Ciencia e Agrotecnologia, 29(6): 1125-1131.
19. Chen, Z.G. and J.F. Wang, 1986. Plantlets derived from early in vitro culture of Citrus zygotic embryo. Journal of Fujian Agricultural University, 15(4):271-276.
20. Chen, C., M.T. Lyon, D. O’Malley, C.T. Federici, J. Gmitter, J.W. Grosser, J.X. Chaparro, M.L. Roose and F.G. Gmitter, 2008. Origin and frequency of 2n gametes in Citrus sinensis × Poncirus trifoliata and their reciprocal crosses. Plant Science, 174:1-8.
21. Cheng, F.S. and M.L. Roose, 1995. Origin and inheritance of dwarfing by the Citrus rootstock Poncirus trifoliata flying dragon. Journal of the American Society for Horticultural Science, 120(2):286-291.
22. Collins, G.B. and J.W. Grosser, 1984. Culture of embryos, cell culture and somatic cell genetics of plants. Laboratory Procedures and Their Applications, 1:241-257.
23. Çevik, B. ve M.Ş. Çevik, 2006. Turunçgillerde genetik mühendisliği uygulamaları. Süleyman Demirel Üniversitesi Ziraat Fakültesi Dergisi, 1(1):74-86.
24. Çimen, B., T. Yeşiloğlu, M. İncesu, B. Yılmaz ve Y.A. Kaçar, 2016. Bazı turunçgil genotiplerinden tetraploid bitki elde edilmesi. Derim, 33(2):175-188.
25. Das, A., A.K. Paul and S. Chaudhuris, 2000. Micropropagation of sweet orange Citrus sinensis Osbeck for the development of nucellar seedling. Indian Journal of Experimental Biology, 38(3): 269-272.
26. De Laat, A.M.M., W. Göhde and M.J.D.C. Vogelzang, 1997. Determination of ploidy of single plants and plant populations by flow cytometry. Plant Breeding, 99:303-307.
27. Doudna, J.A. and E. Charpentier, 2014. Genome editing, the new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213):1258096. https://doi. org/10.1126/science.1258096.
28. Esen, A. and R.K. Soost, 1971. Unexpected triploids in Citrus: their origin, identification and possible use. Journal of Heredity, 62:329-333.
29. Fang, Q., L. Wang, H. Yu, Y. Huang, X. Jiang, X. Deng and Q. Xu, 2018. Plant development of species-specific InDel markers in Citrus. Molecular Biology Reporter (https://doi.org/10.1007/s11105-018-1111-1.
30. Fathi, H. and U. Jahani, 2012. Review of embryo culture in fruit trees. Annals of Biological Research, 3(9):4276-4281.
31. Febres, V.J., C.L. Niblett, R.F. Lee and G.A. Moore, 2003. Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with Citrus Tristeza Closterovirus genes. Plant Cell Reports, 21:421-428.
32. Ferrante, S.P., S. Lucretti, S. Reale, A. De Patrizio, L. Abbate, N. Tusa and M.T. Scarano, 2010. Assessment of the origin of new Citrus tetraploid hybrids by means of SSR markers and PCR based dosage effects. Euphytica, 173:223-233.
33. Friend, W.H. and S.H. Yarnell, 1936. Clonal selection of grapefruit with respect to yield. Proceedings of American Society for Horticultural Science V, 38:358-362.
34. Gamborg, O.L., R.A. Miller and K. Ojima, 1968. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50:151-158.
35. Garcia Lidon, A., 2003. La seleccio'n clonal de Limonero ‘Fino’. Incidencia Del Patro'n. Aspectos Morfolo'Gicos, Agrono'micos y Bioquı'micos. (PhD Thesis, Universidad Polite'cnica de Cartagena).
36. Germana, M.A., 2007. Haploidy. In: I. A. Khan (Ed.): Citrus, Genetics, Breeding and Biotechnology, CABI Publ., pp:167-196.
37. Ghazvini, R.F. and S. Shirani, 2002. Study of the effects of somatic embryogenesis of unfertilized ovules from Mexican Lime (Citrus aurantifolia L.) on different media. Journal of Science and Technology of Agriculture and Natural Resources, 6(2):44-52.
38. Gmitter, F.G., X.B. Ling and X.X. Deng, 1990. Induction of triploid Citrus plants from endosperm cali in vitro. Theoretical and Applied Genetics, 80:785-790.
39. Gmitter, F.G. and X.B. Ling, 1991. Embryogenesis in vitro and nonchimeric tetraploid plant recovery from undeveloped Citrus ovules treated with colchicine. The Journal of the American Society for Horticultural Science, 116(2): 317-321.
40. Göçmen, M., İ. Polat ve Ç. Çakır, 2003. Turunçgil türlerine uygun RAPDs markörlerin belirlenmesi. Derim, 20:43-47.
41. Göral, T., 1987. Turunçgillerde çeşit geliştirme ve olanakları. Derim, 4(2):63-77.
42. Grosser, J.W., F.G. Gmitter and J.L. Chandler, 1988. Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flaying dragon. Plant Cell Reports, 7:5-8.
43. Grosser, J.W., F.G. Gmitter, W.S. Jr. Castle and J.L. Chandler, 1996. Production and evaluation of Citrus somatic hybrid rootstocks. Proceedings of the International Society of Citriculture, 1246-1250.
44. Grosser, J.W., J.L. Chandler and W.L. Duncan, 2007. Production of mandarin + pummelo somatic hybrid Citrus rootstocks with potential for improved tolerance/ resistance to sting nematode. Scientia Horticulturae, 113(1):33-36.
45. Guo, W.W., S.X. Xiao and X. Deng, 2013. Somatic cybrid production via protoplast fusion for Citrus improvement. Scientia Horticulturae, 163:20-26.
46. Gülşen, O. ve N. Mutlu, 2005. Bitki biliminde kullanılan genetik markırlar ve kullanım alanları. Alatarım, 4(2):27-37.
47. Hayashi, K., H. Yoshida and I. Ashikawa, 2006. Development of PCR-based allele-specific InDel marker sets for nine rice blast resistance genes. Theoretical Applied Genetics, 113:251-260.
48. Honsho, C., K. Tsuruta, K. Ryuto, A. Sakata, S. Kuroki, A. Nishiwaki and T. Tetsumura, 2015. Characterization of seed and embryo abortion during fruit development in several Citrus cultivars pollinated by Nishiuchi Konatsu (Citrus tamurana) and preliminary trial of embryo rescue of aborting embryos. Acta Horticulturae, 1065:181-186.
49. Hu, C.Y. and A.G. Ferreira, 1998. Cultura de embrioes. In: Cultura de Tecidos e Transformaçao Genetica de Plantas, 1: 371-393.
50. IAEA, 2018. International Atomic Energy Agency. (https://www.iaea.org).
51. Jaskani, M.J., I.A. Khan and M.M. Khan, 2005. Fruit set, seed development and embryo germination in interploid crosses of Citrus. Scientia Horticulturae, 107:51-57. Y.L., 1993. Recovery and characterization of triploid Citrus hybrids following 2x × 4x hybridization. M.Sc. Thesis, University of California.
52. Jia, H.G. and N. Wang, 2014. Targeted genome editing of sweet orange using Cas9/sgRNA. Plos One, 9(4):e93806.
53. Jia, H., Y. Zhang, V. Orbovic, J. Xu, F.F. White, J.B. Jones and N. Wang, 2017. Genome editing of the disease susceptibility gene CsLOB1 in Citrus confers resistance to citrus canker. Plant Biotechnologie Journal, 15:817-823.
54. Jimenez, V.M., E. Guevara and F. Bangerth, 2001. Endogenous hormone levels in habituated nucellar Citrus callus during the initial stages of regeneration. Plant Cell Reports, 20(1):92-100.
55. Kaneyoshi, J., T. Furuta, T. Shioda, S. Akasaka, Y. Yanagimoto and H. Kurihisa, 2014. Appearance of triploids in natural hybrid seedlings of lemon and breeding of new cultivar ‘Yellow Bell’. The Japanese Society of Horticultural Science, 13:19-26.
56. King, B.J., L.S. Lee and P.T. Scott, 1996. Identification of triploid Citrus by isozyme analysis. Euphytica, 90(2):223-231.
57. Kiong, A.L.P., L.S. Wan, S. Hussein and R. Ibrahim, 2008. Induction of somatic embryos from different explants of Citrus sinensis. Journal of Plant Sciences, 3(1): 18-32.
58. Krug, C.A., 1943. Chromosome numbers in the subfamily Aurantioideae with special reference to the genus Citrus. Botanical Gazette, 104:602-611.
59. Kunitake, H., H. Kagami and M. Mii, 1991. Somatic embryogenesis and plant regeneration from protoplasts of Satsuma mandarin (Citrus unshiu Marc.). Scientia Horticulturae, 47:27-33.
60. Kurt, S. and S. Ulger, 2014. Production of Common Sour Orange × Carrizo Citrange hybrids using embryo rescue. International Journal of Fruit Science, 14:42-48.
61. Kurt, Ş., 2020. Turunçgillerde melezleme yoluyla elde edilen bireylerde uygun embriyo kurtarma aşamasının ve melez bireylerin ploidi seviyesinin belirlenmesi (Doktora Tezi). Isparta Uygulamalı Bilimler Üniversitesi / Lisansüstü Eğitim Enstitüsü, Bahçe Bitkileri Anabilim Dalı, Isparta, 155s.
62. Li, D.D., W. Shi and X.X. Deng, 2002. Agrobacterium-mediated transformation of embryogenic calluses of Ponkan Mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Reports, 21:153-156.
63. Liu, F., G.L. Sun and B. Salomon, 2001. Distribution of allozymic alleles and genetic diversity in the American Barley Core Collection. Theoritical Applied Genetics, 102:606-615.
64. Liu, J.J., K.L. Chen, J. He and B. Guan, 2012. Haploid and polyploid hybrids obtained from cross of diploid Citrus. 12ᵗʰ International Citrus Congress (ICC), Abstract Book, 51.
65. Lu, Y.B., Y.P. Qi, L.T. Yang, J. Lee, P. Guo, X. Ye, M.Y. Jia, M.L. Li and L.S. Chen, 2015. Long-term boron-deficiency-responsive genes revealed by cDNA-AFLP differ between Citrus sinensis roots and leaves. Frontiers in Plant Science, 6:585. (https://doi.org/10.3389/fpls.2015)
66. Luro, F., F. Maddy, P. Ollitrault and D. Rist, 2000. Identification of 2n gamete parental origin and mode of nuclear restitution of spontaneous triploid Citrus hybrids. Proceedings of 9ᵗʰ International Citrus Congress. International Society of Citriculture, 168-169.
67. Matsubara, S., 1964. Effect of nitrogen compounds on the growth of isolated young embryos of Datura. Botanical Magazine, 77:253-259.
68. Maya, M.A., M.G. Rabbani, M.G. Mahboob and Y. Matsubara, 2012. Assessment of genetic relationship among 15 citrus fruits using RAPD. Asian Journal of Biotechnology, 4:30-37.
69. Morais-Leno, L.S., A.S. Souza, W.D.S. Soares-Filho and C.A. Silva-Ledo, 2008. Adjustment of nutrients of the MT medium for the cultivation of Cleopatra tangerine immature embryos. Revista Brasileira de Fruticultura, Jaboticabal SP, 30(1):1-6.
70. Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15:473-497.
71. Murashige, T. and P.H. Tucker, 1969. Growth factor requirements of Citrus tissue culture. Proceedings 1ˢᵗ International Citrus Symposium, 3:1155-1161.
72. Mwangangi, I.M., J.K. Muli and J.O. Neondo, 2019. Plant hybridization as an alternative technique in plant breeding improvement. Asian Journal of Research in Crop Science, 4(1):1-11.
73. Narayanaswamy, S. and K. Norstog, 1964. Plant embryo culture. Botanical Review, 30:587-628.
74. Navarro, L., P. Aleza, J. Cuenca, J. Juárez, J.A. Pina, C. Ortega, A. Navarro and V. Ortega, 2012. The triploid mandarin breeding program in Spain. 12ᵗʰ International Citrus Congress, Abstract Book, 37.
75. Nishimasu, H., F.A. Ran, P.D. Hsu, S. Konermann, S.I. Shehata, N. Dohmae, R. Ishitani, F. Zhang and O. Nureki, 2014. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 156: 935-949.
76. Nito, N. and T. Akihama, 1990. Prospect of Citrus and related genera for disease resistant rootstocks. In: Proceeding IV Asia-Pacific Conference on Citrus Rehabilitation, UNDP-FAO Regional Project RAS/86/022, (pp:39-47), Chiang Mai, Thailand, Hong Kong.
77. Ohgawara, T., S. Kobayashi, K. Ohgawara, H. Uchimiya and S. Ishii, 1985. Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata. Theoretical and Applied Genetics, 71(1):1-4.
78. Ohta, Y. and K. Furusato, 1957. Embryo culture in Citrus. Kihara Institute for Biological Research, 23:49-54.
79. Oiyama, I. and S. Kobayashi, 1990. Poliembryony in undeveloped monoembryonic diploid seeds crossed with Citrus tetraploid. HortScience, 25: 1276-1277.
80. Oliveira, R.P., C.I. Aguilar-Vildoso, C. Mariangela and M.A. Machado, 2004. Skewed RAPD markers in linkage maps of Citrus. Genetics Molecular Biology, 27(3):437-441.
81. Ollitrault, P., D. Dambier, C. Cabasson, C. Teisson and F. Luro, 1994. Protoplast fusion in Citrus. Fruit, 49:5-6.
82. Ollitrault, P., Y. Froelicher, D. Dambier, F. Luro and M. Yamamoto, 2007. Seedlessness and ploidy manipulation. In: I.A. Khan (Ed.): Citrus, Genetics, Breeding and Biotechnology, CABI Publ., pp:197-218.
83. Ollitrault, P., D. Dambier, F. Luro and Y. Froelicher, 2008. Ploidy manipulation for breeding seedless triploid citrus. Plant Breeding Reviews, 20:323-354.
84. Ollitrault, P. and L. Navarro, 2012. Citrus. in: M.L. Badenes, D.H. Byrne (Eds.). Fruit Breeding, Handbook of Plant Breeding (8), Springer Science Business Media, LLC, New York, USA, pp:623-662.
85. Özsan, M., Ö. Tuzcu, Ş.A. Aktepe, H.B.K. Çelikel, E. Özdemir ve İ. Çimen, 1986. Turunçgillerde aşıgözü seleksiyon-sertifikasyonu ve çeşit geliştirme. Derim, 3(3):147-156.
86. Pan, C.H., Z.B. Wang, Y.Y. Ma, Y. Yin, Y.F. Zhang, S.M. Zuo, Z.X. Chen and X.B. Pan, 2008. InDel and SNP markers and their application in mapbased cloning of rice genes. Rice Science, 15:251-258.
87. Pasqual, M., V.G. Riberio and J.D. Ramos, 1990. Influencia do GA₃ e do carvao ativado sobre o enraizamento in vitro de embrioes de laranja Natal. Pesquisa Agropeciaria Brasileira, Brasilia, 25(10):1477-1482.
88. Pasqual, M., D.R. Finotti, L.F. Dutra, E.A. Chagas and L.O. Riberio, 2002a. In vitro culture of Ponca mandarin immature embryos: pH × agar concentration. Revista Brasileira De Agrociencia, 8(3):199-202.
89. Pasqual, M., G.P. Alves, L.F. Dutra, D.R. Finotti and E.A. Chagas, 2002b. In vitro culture of Ponca mandarin immature embryos: MS medium and sucrose concentrations. Revista Ceres, 49(282): 181-189.
90. Pasqual, M., G.P. Alves, L.F. Dutra, E.A. Chagas and L.O. Riberio 2003. In vitro development of immature embryos from Ponca mandarin × Pera sweet orange in different photoperiods. Ciencia e Agrotecnologia, 27(3):535-540.
91. Pena, L., M. Cervera, R. Ghorbel, A. Dominguez, C. Fagoaga, J. Juarez, J.A.and L. Navarro, 2007. Genetic transformation. In: I.A. Khan (Ed.): Citrus, Genetics, Breeding and Biotechnology, CABI Publ., pp:329-344.
92. Perez-Tornero, O. and I. Porras, 2008. Assessment of polyembryony in lemon: Rescue and in vitro culture of immature embryos. Plant Cell Tissue Organ Culture, 93:173-180.
93. Perez-Tornero, O., D. Cordoba, M. Moreno, I. Yuste and I. Porras, 2011. Use of classical methods and tools biotecnologiccas in the genetic improvement of the lemon tree: Preliminary results. Hoticultura Global, 296:14-17.
94. Polat, İ., E. Turgutoğlu and Ş. Kurt, 2015. Determination of genomic diversity within mutant lemon (Citrus limon L.) and mandarin (Citrus reticulata) using molecular markers. Pakistan Journal of Botany, 47:1095-1102.
95. Raghavan, V., 1966. Nutrition, growth and morphogenesis of plant embryos. Biological Reviews, 41:1-58.
96. Raghavan, V., 1976. Experimental embryogenesis in vascular plants. Academic Press, 187-192.
97. Raghavan, V., 2003. One hundred years of zygotic embryo culture investigations. In vitro Cellular and Developmental Biology-Plant, 39:437-442.
98. Raghuvanshi, S.S., 1962. Cytogenetical studies in genus Citrus. IV. Evolution in Genus Citrus, Cytologia, 27:172-188.
99. Rangan, T.S., T. Murashige and W.P. Bitters, 1969. In vitro study of zygotic and nucellar embryogenesis in Citrus. Proceedings of the International Society of Citriculture, 1:225-229.
100. Rao, M.N., J.R. Soneji and L. Sahijram, 2011. Citrus. In: C. Kole (Ed.): Wild Crop Relatives: Genomic and Breeding Resources-Tropical and Subtropical Fruits, Springer, p:43-59.
101. Ray, P.K., 2002. Breeding tropical and subtropical fruits. Springer Science & Business Media, pp:84-106.
102. Reed, S.M., 2005. Embryo rescue. Plant Development and Biotechnology, 18:235-239.
103. Ribeiro, V.G., D. Sanábio, C.N. Souza, P.S.N. Lopes, M.R. Bocardo and M. Pasqual, 2000. Efeitos de acido giberélico e carvão ativado no cultivo in vitro de Citrus limonia Osbeck × Poncirus trifoliata (L.) Raf. Pesquisa Agropecuária Brasileira, 35:27-30.
104. Rietsema, J., S. Satina and A.F. Blakeslee, 1953. The effect of sucrose on the growth of Datura stramonium embryos in vitro. American Journal of Botany, 40:538-545.
105. Roose, M.L., D. Fang, F.S. Cheng, R.I. Tayyar, C.T. Federici and R.S. Kupper, 2000. Mapping the Citrus genome. Acta Horticulturae, 535:25-32.
106. Ruiz, C. and M.J. Asins, 2003. Comparison between Poncirus and Citrus genetic linkage maps. Theoretical and Applied Genetics, 106:826-836.
107. Sahijram, L., J.R. Soneji, A. Naren and B.M. Rao, 2013. Hybrid embryo rescue: a non-conventional breeding strategy in horticultural crops. Journal of Horticultural Science, 8(1):1-20.
108. Sharif, N., M.J. Jaskani and N. Memon, 2013. Responses of Citrus rootstock ovules to colchicine applications in vitro. International Journal of Agricultural Technology, 9(1):201-209.
109. Sharman, D.R. and R.K. Kaur, 1996. Embryo rescue in plants a review. Euphytica, 89:325-337.
110. Soni, A., A.K. Dubey, A. Gupta, R.M. Sharma, O.P. Awasthi, C. Bharadwaj and N. Sharma, 2019. Optimizing embryo age and media for enhancing hybrid seedling recovery in Sour orange (Citrus aurantium) × Sacaton citrumelo (Citrus paradisi × Poncirus trifoliata) crosses through embryo rescue. Plant Breeding, 9p., doi:10.1111/pbr.12690.
111. Soost, R.K. and M.L. Roose, 1996. Citrus. In: J. Janick, J.M. Moore (Eds.): Fruit Breeding, Vol:1, Tree and Tropical Fruits, John Wiley, New York, pp:256-323.
112. Spiegel-Roy, P. and J. Kochba, 1973. Mutation breeding in Citrus. International Atomic Energy Agency (IAEA), Vienna, 91p.
113. Spiegel-Roy, P. and E.E. Goldschmidt, 1996. Biology of Citrus. Cambridge University Press, New York, 230p.
114. Starrantino, A. and F. Russo, 1980. Seedlings from underdeveloped ovules ofripe fruits of poliembryonic Citrus cultivars. HortScience, 15:296-297.
115. Starrantino, A. and G. Reforgiato-Recupero, 1981. Citrus hybrids obtained in vitro from 2x females × 4x males. Proceedings of the International Society of Citriculture, 1:31-32.
116. Swingle, W.T. and P.C. Reece, 1967. The botany of Citrus and its wild relatives. In: W. Reuther, L.D. Batchelor, H.J. Webber (Eds.) The Citrus Industry, University of California; Revised Edition (January 1, 1967), pp:190-340.
117. Sykes, S.R. and W.J. Lewis, 1996. Comparing Imperial mandarin and Silverhill Satsuma mandarin as seed parents in a breeding program aimed at developing new seedless Citrus cultivars for Australia. Australian Journal of Experimental Agriculture, 36:731-738.
118. Şeker, M., O. Tuzcu and P. Ollitrault, 2003. Comparison of nuclear DNA content of Citrus rootstock populations by flow cytometry analysis. Plant Breeding, 122:169-172.
119. Tan, M., J. Song and X. Deng, 2007. Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis. Euphytica, 157:155-160.
120. Taşpınar, M.S. and M. Tosun, 2002. İzoenzim elektroforez tekniğinin bitki ıslahında kullanımı. Atatürk Üniversitesi Ziraat Fakültesi Dergisi, 33(4):451-456.
121. Thao, N.T.P., K. Ureshino, I. Miyajima, Y. Ozaki and H. Okubo, 2003. Induction of tetraploids in ornamental alocasia through colchicine and oryzalin treatments. Plant Cell Tissue Organ Culture, 72:19-25.
122. Thuy, H.T., D.N. Vinh, T.T. Hanh, T.N. Thanh, L.Q. Hung, V.A. Tuan and T.D. Khanh, 2014. Integration of biotechnology and conventional breeding to develop Citrus seedless cultivars in Vietnam. International Journal of Development Research, 4(10):2091-2093.
123. Tuna, M., 2016. Flow sitometri ve tarımsal araştırmalarda kullanımı. IV. Flow Sitometri ve Tarımsal Araştırmalarda Kullanımı Uygulamalı Eğitim Programı, 29-30 Ocak, Tekirdağ, 1-24.
124. Turgutoğlu, E., Ş. Kurt, G. Demir ve Z. Eryılmaz, 2012. BATEM’de geliştirilen yeni turunçgil çeşitleri. Borsanomi Dergisi, 35:56-58.
125. Tusa, N., S. Fatta Del Bosco, L. Nardi and S. Lucretti, 1996. Obtaining triploid plants by crossing Citrus lemon cv. Femminello 2n × 4n allotetraploid somatic hybrids. Proceedings of the International Society of Citriculture 1:133-136.
126. Tuzcu, Ö., M. Kaplankıran ve T. Yeşiloğlu, 1988. Turunçgillerde radyasyon uygulaması ile yeni çeşitlerin ıslahı. Çukurova Üniversitesi Araştırma Fonu 1. Bilim Kongresi Bildirileri, 1:25-34.
127. Tuzcu, Ö., B. Yıldırım, S. Düzenoğlu ve İ. Bahçeci, 1999. Değişik turunçgil anaçlarının Washington Navel ve Moro kan portakal çeşitlerinin verim ve kalitesi üzerine etkileri. Journal of Agriculture and Forestry, 23:213-222.
128. Ulubelde, M., 1989. Turunçgillerde mutasyon. Derim, 6(1):38-48. Umbeck, P.F. and K. Norstog, 1979. Effects of abscisic acid and ammonium ion on morphogenesis of cultured barley embryos. Bulletin of the Torrey Botanical Club, 106:110-116.
129. Usman, M., M. Ramzan, B. Fatıma, M.J. Jaskani and M.M. Khan, 2002. Citrus germplasm enhancement by interploid hybridization. International Journal of Agriculture and Biological Sciences, 4(2): 208-210.
130. Usman, M., B. Fatima, W. Abdus Samad and K. Bakhsh, 2012. Embryo culture to enhance efficiency of colchicine induced polyploidization in grapefruit. Pakistan Journal of Botany, 44:399-405.
131. Uzun, A., O. Gülşen, G. Kafa and U. Seday, 2008. Alata, Gulsen and Uzun seedless lemons and Eylul early-maturing lemon. Hortscience, 43:1920-1921.
132. Uzun, A., 2009. Turunçgillerde genetik çeşitliliğin SRAP markırları ile karakterizasyonu (Doktora Tezi). Çukurova Üniversitesi Fen Bilimleri Enstitüsü, Bahçe Bitkileri Anabilim Dalı, Adana, 384s.
133. Vali, U., M. Brandstrom, M. Johansson and H. Ellegren, 2008. Insertion-deletion polymorphisms (InDels) as genetic markers in natural populations. BMC Genetics, 9(1):8. doi:10.1186/1471-2156-9-8.
134. Veen, H., 1963. The effect of various growth-regulators on embryos of Capsella bursapastoris growing in vitro. Acta Botanica Neerlandica, 12:129-171.
135. Viloria, Z., J.W. Grosser and B. Bracho, 2005. Immature embryo rescue, culture and seedling development of acid Citrus fruit derived from interploid hybridization. Plant Cell, Tissue and Organ Culture, 82:159-167.
136. Wakana, A., X.N. Binh and M. Iwamasa, 2004. Germinability of embryos during seed development in Citrus (Rutaceae). Journal of the Faculty of Agriculture, Kyushu University, 49(1):49-59.
137. Wang, J.F., Z.G. Chen and T.X. Lin, 1999. Observation on the embryonic development in Citrus after cross pollination. Chinese Developmental Reproductive Biology Society, 8(2):57-63.
138. White, P.R., 1934. Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiology, 9:585-600.
139. Williams, E.G., I.M. Verry and W.M. Williams, 1982. Use of embryo culture in interspecific hybridization. In: Plant Improvement and Somatic Cell Genetics, pp:119-128.
140. Xie, K.D., X.P. Wang, M.K. Biswas, W.J. Liang, Q. Xu, J.W. Grosser and W.W. Guo, 2014. 2n megagametophyte formed via SDR contributes to tetraploidization in polyembryonic ‘Nadorcott’ tangor crossed by Citrus allotetraploids. Plant Cell Reports, 33:1641-1650.
141. Xie, K.D., D.Y. Yuan, W. Wang, Q.M. Xia, X.M. Wu, C.W. Chen, C.L. Chen, J.W. Grosser and W.W. Guo, 2019. Citrus triploid recovery based on 2x × 4x crosses via an optimized embryo rescue approach. Scientia Horticulturae, 252:104-109.
142. Yeoman, M.M., 1986. Plant cell culture technology. Blackwell Scientific Publisher, Biotanical Monographs, 23.
143. Yeşiloğlu, T., 2017. Turunçgil anaçlarının tarihçesi ve yeni anaçların geliştirilmesi. Türkiye Tohumcular Birliği Dergisi, 22:12-14.
144. Yeung, E.C., T.A. Thorpe and C.J. Jensen, 1981. In vitro fertilization and embryo culture. In: T.A. Thorpe (ed.): Plant Tissue Culture: Methods and Applications in Agriculture, Academic Press, pp:253-271.
145. Yi, H., X. Deng and C. Fu, 2001. Application of embryo rescue techniques in fruit crops. Journal of Fruit Science, 18(4):224-228.
146. Zdrujkovskaja-Richter, A.I., 1981. Embryo cultures and development of new forms of plants. University Moscow, Abstract.
147. Zhang, F., C. LeBlanc, V. Irish and Y. Jacob, 2017. Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter. Plant Cell Reports, 36(12):1883-1887.
148. Zhu, S., B. Wu, Y. Ma, J. Chen and G. Zhong, 2013. Obtaining Citrus hybrids by in vitro culture of embryos from mature seeds and early identification of hybrid seedlings by allele-specific PCR. Scientia Horticulturae, 161:300-305.