In Vitro Production of Autotetraploid Ponkan Mandarin (Citrus Reticulata Blanco) Using Cell Suspension Cultures
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Euphytica (2010) 173:235–242 DOI 10.1007/s10681-009-0098-y In vitro production of autotetraploid Ponkan mandarin (Citrus reticulata Blanco) using cell suspension cultures M. Dutt • M. Vasconcellos • K. J. Song • F. G. Gmitter Jr. • J. W. Grosser Received: 10 March 2009 / Accepted: 7 December 2009 / Published online: 22 December 2009 Ó Springer Science+Business Media B.V. 2009 Abstract An efficient protocol for colchicine med- Keywords Autotetraploid Á Cell suspension Á iated production of in vitro autotetetraploids from Citrus reticulata Á Colchicine Á Ponkan mandarin using cell suspension cultures is Enzymatic maceration Á Flow cytometry described. Cells were treated with 1 g l-1 colchicine for 4 or 8 days before transfer into solid EME Abbreviations medium supplemented with 5% maltose. Colchicine DAPI 40,6-Diamidino-2-phenylindole treated cells were placed in medium with or without MT Murashige and Tucker an overlay of 1:2 medium–mixture of liquid 0.6 M UV Ultraviolet BH3 medium and 0.15 M EME ? maltose liquid media. It was observed that modifying the immediate cell environment by addition of the liquid overlay played a positive role in cell differentiation and Introduction subsequent plant regeneration. Ploidy levels were determined with a flow cytometer and confirmed by Ponkan mandarin (Citrus reticulata Blanco) is one of chromosome staining using the enzymatic maceration the most important commercial mandarin cultivars, method. A large number of non-chimeric autotetrap- cultivated extensively in many countries of the world loids were generated using this method. Such plants of which China and Brazil are the major producers. have great value in a breeding program for the Although the fruit is sweet and easy to peel, it is development of seedless triploid citrus, as very few seedy, which makes it less consumer friendly (Li available tetraploid breeding parents are easy to peel. et al. 2002). In recent years, there has been a shift in the world citrus market towards seedless citrus fruits and considerable energy has been devoted towards M. Dutt Á M. Vasconcellos Á K. J. Song Á their production. The seedless trait in citrus is related F. G. Gmitter Jr. Á J. W. Grosser (&) to male or female gametophyte sterility, self incom- Horticultural Sciences Department, Citrus Research patibility, or early embryo abortion (Reforgiato and Education Center, University of Florida-IFAS, 700 Experiment Station Road, Lake Alfred, Recupero et al. 2005), and several methods exist for Fl 33850, USA the production of seedless citrus of which mutation e-mail: jgrosser@ufl.edu breeding, somaclonal variation and triploid breeding are the most important. Of the three, triploidy has K. J. Song Faculty of Bioscience and Industry, Cheju National been successfully utilized in many cultivars like University, 66 Jejudaehakno, Jeju 690-756, Korea banana, watermelon and grapes for the production of 123 236 Euphytica (2010) 173:235–242 seedless fruits. In citrus, triploid seedless cultivars are cells which in turn would result in stable, non- obtained by breeding between elite monoembryonic chimeric tetraploid plants due to the single cell origin diploid cultivars as female parent with tetraploid of somatic embryos (Stewart et al. 1958). In citrus, cultivars as pollen parent (Esen and Soost 1973). autotetraploid plants induced by in vitro treatment of Sterility in such fruits is caused due to the odd embryogenic callus with colchicine resulted in the number of chromosomes that are unable to undergo production of autotetraploid plants (Wu and Mooney successful meiotic pairing to produce chromosomally 2002). However, the frequency was low. Recently, balanced gametes (Reforgiato Recupero et al. 2005). Zhang et al. (2007) described a protocol to obtain In the past this strategy has been hampered due to the autotetraploid citrus plants using callus and generated unavailability of superior tetraploid parents (Ollitrault a number of sweet orange plants. et al. 2000). However, in recent years the pool of We describe herein a simple and efficient protocol superior tetraploid breeding parents has been for the production of autotetraploid plants at a high enriched due to the production of tetraploid cultivars frequency from the mandarin cultivar ‘Ponkan’ using using colchicine (Gmitter and Ling 1991; Gmitter cell suspension cultures. We also detail incorporation et al. 1991; Juarez et al. 2004) and heterozygous of a liquid overlay of a nutrient rich medium for rapid tetraploid cultivars obtained through somatic hybrid- development of embryoids. ization (Grosser and Gmitter 1990; Grosser et al. 1992, 2000; Grosser and Chandler 2004; Grosser and Gmitter 2005). Materials and methods Colchicine is an alkaloid obtained from the meadow saffron (Colchicum autumnale L.). This Production of suspension cells alkaloid inhibits mitosis by hampering the develop- ment of the nuclear spindle (Blakeslee and Avery Immature fruit (approximately 150 days after pollina- 1937) and is most commonly used to obtain tetraploid tion) were collected from a single ‘Ponkan’ mandarin plants artificially (Notsuka et al. 2000). Chromo- tree maintained at the Citrus Research and Education somes have been doubled in several fruit crops such Center campus, Lake Alfred, Florida. Fruits were as banana (Hamill et al. 1992), blueberry (Lyrene and surface sterilized for 20 min in a 0.6% (v/v) sodium Perry 1982), cherry (James et al. 1987) and grapes hypochlorite solution containing a drop of Tween 20 (Notsuka et al. 2000) using colchicine. and rinsed 39 with sterile deionized water. Unfertil- In citrus, tetraploidy has been induced by treat- ized ovules were extracted from these fruits and plated ment of axillary buds with colchicine, as was done onto solid callus induction medium (DOG medium with the cultivars Ellendale and Clementine. The consisting of MT (Murashige and Tucker 1969) salts treated buds upon grafting on rootstock produced and vitamins supplemented with 50 g l-1 sucrose, several tetraploid plants (Oiyama 1992). However, a 0.50 g l-1 malt extract, 8 g l-1 agar and 5 mg l-1 disadvantage of using axillary buds in colchicine kinetin). Ovules were subcultured on a monthly basis experiments is that most of the recovered plants end until production of embryogenic callus. The callus was up being unstable chimeras and do not have appli- maintained by monthly transfer on a hormone free cations in a breeding program (Barrett 1974; Jaskani callus-maintenance (EME) medium (Grosser and et al. 1996). This is due to the use of multicellular Gmitter 1990). For cell suspension culture, approxi- tissue as a source of explants for colchicine treatment. mately 5 g of callus was incubated in 25 ml liquid Using such tissues usually result in production of a H ? H cell proliferation medium on a 2 week transfer large proportion of chimeric tetraploids (Kadota and cycle according to Grosser and Gmitter (1990). Niimi 2002). Non-chimeric autotetraploid citrus Actively dividing suspension cells were treated with plants have been obtained from in vitro colchicine colchicine 7 days after the third subculture. experiments via embryogenesis of underdeveloped ovules from immature citrus fruits (Gmitter and Ling Colchicine treatment 1991; Gmitter et al. 1991). The use of cell suspension cultures offers the Colchicine (Sigma-Aldrich Corp. USA) was dis- possibility to produce single tetraploid embryogenic solved in a few drops of dimethylsulfoxide (DMSO) 123 Euphytica (2010) 173:235–242 237 and volume made up with sterile water to a final instructions provided on a CyStain UV precise P kit concentration of 1 g ml-1. The solution was filter (Partec). The position of the 2n peak was determined sterilized and stored at -20°C until use. Approxi- from nuclear DNA obtained from a known diploid mately 1 g of cells were harvested and placed in fresh standard on the machine’s histogram. liquid H ? H medium supplemented with 1 g l-1 colchicine. The cells were incubated on a platform Chromosome preparation and staining shaker at 30 rpm for 4 or 8 days. Chromosome preparation and staining were per- Induction of polyploid plantlets and determination formed as described by Yahata et al. (2006) with of ploidy modifications. Young leaves (approximately 3–5 mm long) were excised from plants obtained by colchi- After incubation with colchicine for 4 and 8 days, cine treatment, immersed in 2 mM 8-hydroxyquino- suspension cells were washed twice with H ? H line at 10°C for 12 h in dark, and fixed in methanol– medium and plated on solid EME medium supple- acetic acid (3:1). The fixed leaves were digested with mented with 50 g l-1 maltose (henceforth called EME) an enzyme mixture containing 2% Cellulase Onozuka for proliferation. The cultures were maintained at 28°C RS (Phytotechnology Lab., USA), 1% Macerozyme with a standard 16 h light/8 h dark cycle using cool R-10 (Research Products International Corp., USA) white fluorescent lights (75 lmol s-1 m-2). Two and 0.3% Pectolyase Y-23 (MP Biomedicals, LLC, treatments were carried out. In the first, the cells were USA) in hypotonic solution (75 mM KCl, 7.5 mM plated solely onto EME medium, while in the second Na2-EDTA) at 37°C for 1 h. The digested samples the cells plated on EME medium were overlaid with were transferred to slide glasses, smeared with a drop 2 ml of a 1:2 (v:v) mixture of 0.6 M BH3 medium and of fixative solution using a fine-pointed forceps, and 0.15 M EME ? maltose liquid medium, as utilized in air dried. Chromosomes were stained with a 2% our citrus protoplast regeneration protocol (Grosser Giemsa solution (Merck Co., Germany) in phosphate and Gmitter 1990). For each treatment, the colchicine buffer (pH 6.8) for 45 min, rinsed with distilled treated cells were equally divided and plated into 6 water, air dried, observed and photographed under an individual plates, and each plate was considered as a optical microscope.