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 Cell suspension iated production of in vitro autotetetraploids from Citrus reticulata Colchicine Ponkan mandarin using cell suspension cultures is Enzymatic maceration 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 M. Vasconcellos K. J. Song their production. The seedless trait in citrus is related F. G. Gmitter Jr. 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. replicate. Experiments were repeated twice. Regenerated embryoids were cultured over 0.22 mm cellulose acetate membrane filters placed Results and discussion on solid EME medium to normalize and enlarge the embryoids (Niedz et al. 2002). Somatic embryos were Effect of colchicine on development of somatic subsequently enlarged on EME 1500 embryo matu- embryos ration medium and germinated on B? medium. Ploidy was tested from the first true leaf of the Actively dividing suspension cells were placed in geminating plantlet. Plantlets observed to be tetra- liquid H ? H cell proliferation medium supple- ploid were transferred for rooting and growth into mented with 1 g l-1 colchicine. Cells to be treated RMAN rooting medium. After a month of growth in with colchicine were harvested 7 days after the third vitro the rooted plantlets were potted into metromix suspension subculture. It has been found that a commercial potting medium and acclimated to majority of citrus cells are in S-phase of mitotic cycle greenhouse conditions. Unless otherwise mentioned, on days 6 and 7 from the beginning of their all media formulations were as described by Grosser subculture (Zhang et al. 2007; our observations). and Gmitter (1990). This phase corresponds to the stage when DNA Ploidy analysis was performed with a tabletop synthesis and replication occurs and is ideal for Ploidy Analyser flow cytometer (Partec GmbH, application of colchicine since this antimitotic agent Germany). A small piece (approximately 0.4 cm2) can only act on actively dividing cells (Zhang et al. of leaf was chopped with a sharp blade in extraction 2007). H ? H medium has been widely used buffer, passed through a 45 lm nylon mesh screen, (Grosser and Gmitter 1990, 2005) and in our studies and stained with fluorescent dye (DAPI) as per was considered to be the best medium for 123 238 Euphytica (2010) 173:235–242 proliferation of suspension cells. Use of a liquid osmoticum in a developing seed promotes a cascade medium also has advantages over solid medium due of events that are necessary for embryogenesis to to absence of gradients of nutrients in the medium. occur (Brisibe et al. 1994). Osmoticum also plays a After treatment with colchicine, suspension cells role in obtaining correct storage protein expression in were plated in EME medium. developing somatic embryos (Merkel et al. 1995). We It was observed that the time required for prolif- used 1:2 medium–mixture, with its high osmotic eration of cells was dependent on the time of levels, to mimic the process of development of an treatment with colchicine. Four day treated cells embryo in the seed. This medium also supplemented plated on EME medium supplemented with 1:2 liquid the basal EME solid medium and provided essential medium–mixture (0.6 M BH3: 0.15 M EME ? malt- nutrients necessary for rapid cell growth and division ose) were quicker to proliferate into embryogenic and promoted embryogenesis. This resulted in callus that those without the addition of any supple- enhanced embryo production over treatments which ment. There was no difference in the regeneration did not contain the 1:2 medium–mixture overlay capacity of 8 day treated cells with or without the (Table 1). Subsequent transfer to an embryo matura- addition of 1:2 medium–mixture as most cells did not tion medium brought down osmoticum levels and proliferate in either treatments (Table 1). During the allowed normal germination to occur, since continued first 2 weeks of culture, suspension cells treated for high osmotic levels usually result in inhibition of 8 days with colchicine also turned dark brown while embryo germination by blocking water uptake (Fin- ones treated for 4 days did not. BH3 medium is based kelstein and Crouch 1986). on a formulation described by Kao and Michayluck Untreated suspension cells proliferated into (1975) and modified by Grosser and Gmitter (1990) embryogenic calli within 4 weeks of plating, while for citrus. This medium contains most nutritional 4 day colchicine treated cells required an additional 2 requirements for growth of citrus cells and has been weeks to proliferate when overlaid with the 1:2 liquid used extensively in protoplast fusion experiments medium–mixture (Table 1). The establishment of (Grosser and Gmitter 1990) while EME medium embryogenic cells was followed by the typical supplemented with maltose is efficient in inducing globular, torpedo and cotyledonary states of somatic somatic embryogenesis as observed on nucellar callus embryos from 9th to 13th week after colchicine of orange (Vardi and Galun 1988). treatment. Small translucent and globular somatic In developing seeds, higher osmotic levels have embryos were visible on the callus after 2 passages of been observed to be an important factor associated the callus into fresh EME medium, approximately with embryogenesis and storage reserve accumula- after 10 weeks of treatment with colchicine (Fig. 1). tion (Fujii et al. 1990; Attree et al. 1992). Higher Individual embryos were transferred into EME 1500 maturation medium, where embryos in the globular Table 1 Effects of the addition of colchicine on the regener- and torpedo stage matured into the cotyledonary ation potential of Ponkan cell suspension cultures stage and embryos in cotyledonary stage initiated Treatment Time for callus/embryo Average embryo shoot and root primordium. Somatic embryos also (days) production (weeks)a production ± SEb developed quicker in medium with the 1:2 medium– mixture overlay (Table 1). However, a large percent- 0 4/6 175 ± 35 age of embryos that developed on the 8 day treatment 4 7/10 65.5 ± 10 were misshapen without any visible root or shoot 4 (1:2) 6/9 110.3 ± 22 primordium. A majority of embryos originating from 8 12/16 10.1 ± 2 the 8 day treatment failed to advance from the 8 (1:2) 12/16 18.8 ± 10 globular stage into the torpedo stages despite several a Time for callus production is the time required for initiation passages on the EME 1500 medium. These embryos of fresh callus cells. Time to embryo production was recorded also did not germinate when placed on B? germina- as time required for the production of the first visible globular stage embryo tion medium. Colchicine, as mentioned earlier, is a b Number of embryos produced after 4 weeks of initiation of toxic antimitotic agent which functions by disrupting the first globular stage embryo. Data was recorded from 6 microtubule formation and the differentiation pro- individual plates, each treated as a replicate cess. In general, higher concentration and longer 123 Euphytica (2010) 173:235–242 239

Fig. 1 Steps in the generation of tetraploid Ponkan mandarin: a embryogenic callus, b embryos regenerating from colchicine treated cell suspension cultures, c globular stage embryos developing on acetate paper in EME medium, d putative tetraploid plantlets germinating in B? medium, e tetraploid plantlets in RMAN medium, f tetraploid plant acclimatized under greenhouse conditions

duration of treatment leads to a reduction in survival However, after 8 days of treatment with colchicine, and germination of embryos. Chromosome doubling even the 1:2 medium–mixture overlay did not have in vitro using colchicine also depends on a balance an impact on tetraploid plant production (Table 2), between toxicity of the chemical and genome dou- suggesting that longer duration reduced survival and bling efficiency (Grzebelus and Adamus 2004). A subsequent proliferation of suspension cells. We did high concentration or a long duration of treatment not identify any chimeric plant from the different usually results in the formation of very few plants as treatments. This confirmed that somatic embryogen- a result of toxicity (Hansen et al. 2000). In our esis, due to its single cell origin, decreased consid- experiments, untreated cells developed embryos erably the possibility of chimeric plant production. within 6 weeks of transfer, while treated cells No tetraploid plantlets were obtained in treatments required 9 or more weeks to initiate embryogenesis. regenerated from untreated embryos. Tetraploid cells have been observed to occur spontaneously in many Determination of ploidy callus lines after several passages in medium. For example, in 10 year old ‘Murcott’ tangor callus Depending on growth, germinating embryos in B? maintained through regular subcultures, 4.4% of the medium were evaluated for ploidy and transferred to total cells were observed to be tetraploid in nature RMAN rooting medium. Several embryos failed to (Grosser et al. 2007). In theory, tetraploid plants can develop further and these were transferred into fresh be obtained from such callus lines, but in practice medium until shoots developed. Individual plantlets would be difficult to regenerate successfully, given were described as diploid or tetraploid according to the age of the callus. The callus line used in our the peaks obtained by flow cytometry. Representative experiment was a vigorous, young line (less than examples are illustrated in Fig. 2. Our initial hypoth- 6 months of age), and we did not observe any esis, that overlaying colchicine treated cells with 1:2 spontaneous mutations in the control. medium–mixture would have a positive impact on the The ploidy level of plants that had been observed regeneration of tetraploid cells was validated. The to be tetraploid was further confirmed by chromo- majority of tetraploid plants were obtained from the some staining. Identification of citrus chromosomes 4 day colchicine treatment that included the liquid has been difficult because of their small size 1:2 medium–mixture overlay (34.7%). Compara- (Yamamoto et al. 2008). However, by application tively, the 4 day treatment without the liquid overlay of the enzymatic maceration method (Kurata and produced 10.7% tetraploid plants and this decreased Omura 1978; Yahata et al. 2006), it is relatively with increase in treatment time with colchicine. easier to isolate chromosomes from young leaves 123 240 Euphytica (2010) 173:235–242

Fig. 2 Representative histograms obtained with a flow cytometer exhibiting: a diploid and b tetraploid plant

when compared to staining of root chromosomes (our Table 2 Influence of colchicine treatments on generation of tetraploid Ponkan seedlings. Embryogenic cell suspension observations). Chromosome staining results sup- cultures were treated with 1 g l-1 colchicine in liquid H ? H ported the data obtained using the flow cytometer medium before transfer to solid EME medium and we observed 2n = 18 in control plants and Treatment No of germinating Tetraploid Tetraploid 4n = 36 in analyzed tetraploid plants (Fig. 3). (days) seedling tested (No.) (%)

0 75 0 0.0 Conclusion 4 75 8 10.7 4 (1:2) 75 26 34.7 Our results demonstrated the ability to produce a 8 75 4 5.3 large number of non-chimeric tetraploid plants from 8 (1:2) 75 2 2.7 cell suspension cultures using simple modifications to

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Fig. 3 Visualization of stained chromosomes following enzymatic digestion of leaf cells. a Diploid (2n = 18) and b tetraploid (4n = 36)

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