Somatic hybridization in order to obtain tetraploid parents for citrus scion improvement

Author(s)

Leonardo Soriano

• Document type: Master's Dissertation
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2011-01-27

Examining board members

Francisco de Assis Alves Mourão Filho; Maria Angélica Pereira de Carvalho Costa; Mariangela Cristofani Yaly

• Advisor: Beatriz Madalena Januzzi Mendes

Abstract

Somatic hybridization by protoplasts fusion is a method of genetic combination of two parental genomes, and their cytoplasmic organelles. It is an alternative tool to conventional breeding methods. The objective of this work was to obtain interspecific somatic hybrids of \'Pera\' and \'Westin\' sweet oranges (Citrus sinensis L. Osbeck) with \'Fremont\' (C. clementina hort. ex Tan. x C. reticulata Blanco), \'Thomas\' (C. reticulata Blanco) and \'Nules\' (C. clementina hort. ex Tan) mandarins and Nova tangelo (C. reticulata Blanco x C. paradisi Macf.) to produce tetraploid parents with desirable agronomic characteristics. The sources of protoplasts were sweet orange embryogenic callus and mandarin and tangelo young leaves, collected from plants cultivated in screenhouses. For protoplasts isolation, cells from both sources were immersed in enzyme solution and incubated in the dark for 14 hours (40 rpm). After isolation, the protoplasts isolated from callus were fused chemically (polyethylene glycol - PEG) with non embryogenic protoplasts, isolated from leaf mesophyll. The protoplasts were cultivated in BH3, BH3 + EME and EME liquid culture media and incubated in the dark, under temperature of 27 ºC. After 20 days of incubation, subcultures were initiated in order to reduce the osmotic potential of culture medium. The microcalus developed were transferred to EME medium supplemented with maltose (13 g.L-1) to induce somatic embryogenesis. The developed embryos were transferred into EME + sucrose (25 g.L- 1), culture medium 1500 (1.5 g.L-1 of malt extract) or B+ culture medium for development and germination. The regenerated plants were individually rooted or micrografted, and further acclimatized in screenhouse. Somatic hybridization was confirmed by analysis of leaf morphology, ploidy determination by flow cytometry and molecular analysis by SSR and RAPD markers. Somatic hybrids were obtained of the three combinations (Pera + Fremont, Pêra + Nules and Pêra + Nova) which will be evaluated for direct are as scion or as a tetraploid parent in citrus breeding programs. (AU)

• FAPESP's process: 08/07446-6 - Somatic hybridization aiming the production of tetraploides parents usefull for scion cultivars breeding.
• Grantee: Leonardo Soriano
• Support Opportunities: Scholarships in Brazil - Master