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Colchicine-induced stable autotetraploid plants were recovered through indirect organogenesis from stem sections of in vitro-grown zygotic seedlings of pink/red-fleshed pummelo (Citrus grandis L. Osbeck) selections cybrid Hirado Buntan (C-HBP) (pink), 5-1-99-3 (dark pink), and HBJL-5 (red), all derived from Hirado Buntan pink pummelo. Multiple shoot induction was achieved through indirect organogenesis from the callus produced from the cut ends of the treated explants. Ploidy levels of regenerated plantlets were determined through flow cytometry at a stage when recovered shoots had one to two expanded leaves. Recovered tetraploids proved to be stable after 2 years in the field. As expected, higher colchicine concentrations and treatment durations decreased the survival rates of the regenerated plantlets. Colchicine concentrations of 0.05% and 0.1% produced the most tetraploids; of the 19 total tetraploids produced, 10 were produced from the treatment with a colchicine concentration of 0.1% and six were produced from treatment with a concentration of 0.05%. After flowering, these stable pink/red-fleshed tetraploid plants generated should be useful as breeding parents in our grapefruit/pummelo improvement program, especially if any show canker tolerance or reduced furanocoumarins. Use of monoembryonic tetraploids in interploid citrus crosses eliminates the need for embryo rescue to recover seedless triploid progeny; this research expands our pool of available high-quality monoembryonic tetraploid parents. Grapefruit (Citrus paradisi Macf.) is an important crop in Florida and is grown for fresh fruit and juice. Florida contributes almost 30% of the world grapefruit production. Grapefruit is believed to have originated in Barbados as an accidental cross between a pummelo (C. grandis Osbeck) and a sweet orange [Citrus sinensis (L.) Osbeck] (Barrett and Rhodes, 1976; Gmitter, 1995; Scora, 1975; Scora et al., 1982). The increasing competition and popularity of grapefruit in international markets have stimulated grapefruit breeders to develop new varieties that can meet consumers’ demands. Grapefruit vary in flesh color with white, pink, and red cultivars being available. The pinkand red-fleshed varieties are most popular and earn higher prices in the markets. Another highly desired trait in commercial grapefruit cultivars these days is seedlessness. Seedless varieties of fruits like banana, watermelon, grapes, and plantain are available in the market and sell more than their seedy counterparts. Seedlessness has gained importance in citrus in the recent past. Seediness is causing problems in the acceptance of the fruit in the local or international markets and can even act as a barrier toward release of a variety. Breeders worldwide are trying to generate seedless cultivars with improved quality and disease resistance. Seedlessness has been achieved in the past through approaches ranging from traditional hybridization to biotechnology. Seedlessness has been long associated with triploids. Seedless triploids have been selected from spontaneously occurring triploids in natural populations (Geraci et al., 1975; Wakana et al., 1982), from somaclonal variation (Deng et al., 1985), from diploid · diploid crosses (Esen and Soost, 1971; Geraci, 1978; Geraci et al., 1977), somatic hybridization between haploid and diploid parents (Kobayashi et al., 1997), from endosperm culture (Chen et al., 1991; Wang and Chang, 1978), from genetic transformation (Koltunow et al., 1998), and from interploidal hybridization between a tetraploid and a diploid (Esen and Soost, 1972; Grosser and Gmitter, 2011). Interploidal hybridization is the most common and efficient way for breeders to generate triploid cultivars. However, triploid breeding programs always face a shortage of quality tetraploids for use as parents in such crosses. Crosses where tetraploids are used as female parents are more efficient and have much higher triploid recovery than the reciprocal cross. This is the result of normal fertilization between female diploid and male haploid gametes (Cameron and Burnett, 1978; Esen and Soost, 1972; Soost and Cameron, 1975). However, when the tetraploid female parent is a polyembryonic cultivar, the hybrid embryo needs to be rescued under sterile conditions and has to be grown in vitro. This is performed to avoid the suppression of the zygotic hybrid triploid embryo by the dominating nucellar embryos that are present. This technique is not costor labor-effective and lowers the effectiveness of triploid breeding programs. On the other hand, use of a tetraploid monoembryonic selection as a female parent in such crosses eliminates the need for embryo rescue and hybrid progeny can be easily recovered. We have already generated several hundred triploid grapefruit-like hybrids using this approach (J.W. Grosser, unpublished data) using only a few available monoembryonic tetraploid parents. One approach to overcome the limitation polyembryony imparts is to use the pummelo gene pool in grapefruit breeding. The pummelo is an ideal candidate for development of new grapefruit cultivars because it is one of the ancestors of grapefruit, is a true species, and introduces genetic diversity. Pummelo is monoembryonic and would eliminate the need for embryo rescue when used in interploidal crosses as a female parent. A large range of red-fleshed pummelo selections is available in the Citrus Research and Education Center, Lake Alfred, FL (CREC) germplasm collection that can be used as potential parents. Pummelos can have lower quantities of undesirable compounds such as naringin and furanocoumarins. Thus, when used in crosses, it should be possible to generate desirable seedless hybrids with reduced levels of these compounds. Colchicine is an alkaloid obtained from Colchicum autumnale, which acts as a mitotic inhibitor (Blakeslee and Avery, 1937) and induces tetraploidy in the target cells by interfering with spindle formation at the metaphase. Colchicine is commonly used to induce tetraploidy in breeding lines in Citrus. Early attempts to generate autotetraploids by treating the axillary buds with colchicine ex vitro were performed by Barrett (1974) in monoembryonic cultivars. However, his technique did not produce any non-chimeric tetraploid plants. Later work showed that autotetraploids can be produced in monoembryonic cultivars from colchicine treatment of axillary buds in vitro (Oiyama and Okudai, 1986) or somatic embryogenic callus (Wu and Mooney, 2002). Recently, we reported on the production of autotetraploid pummelo plants through colchicine treatment of germinating seed (Kainth and Grosser, 2010). However, we are aware of no other reports of tetraploid induction in pummelo at present. This study reports a more efficient method to induce tetraploids by in vitro treatment of cut stem explants from pummelo selections with colchicine followed by shoot induction through indirect organogenesis. The effect of different colchicine concentrations and exposure durations was compared for efficiency of indirect organogenesis and tetraploid induction. Received for publication 17 Sept. 2013. Accepted for publication 6 May 2014. To whom reprint request should be addressed; e-mail manjul@ufl.edu. 944 HORTSCIENCE VOL. 49(7) JULY 2014 Materials and Methods Plant materials. Pummelo selections 5-199-3, C-HBP, HBJL-5, and HBJL-7 were selected for this study on the basis of their pink or red flesh and other quality characteristics. These selections are all derived from the ‘Hirado Buntan’ pink pummelo and are located at the experimental farm of the CREC. The seeds were extracted from the mature fruit of each selection, treated with sodium hydroxide for 4 to 5 min for slime removal, and rinsed three to four times with water. The seeds were dried and refrigerated at 4 C until used. For this experiment, seeds from each selection were peeled and sterilized with 10% Clorox bleach (6.25% sodium hypochlorite) and two drops of Tween-20. Individual seed were put into 15-cm long test tubes containing 15 mL of solid Murashige and Skoog (MS) medium consisting of MS salts and vitamins (Murashige and Skoog, 1962) supplemented with 30 g·L sucrose and 7 g·L agar, pH 5.8. These were then placed in the dark for 4 weeks. The etiolated conditions were provided to facilitate and hasten stem elongation and suppress lateral branching. At 4 weeks, when the seedlings attained a height of 12 to 15 cm, they were put under continuous light conditions for 4 to 5 d to allow greening and hardening of the stems. At this time, the seedlings were ready for colchicine treatments. Colchicine treatments. After hardening of the seedlings, the leaves were removed and each stem was cut into 1-cm long explants by making a diagonal cut at both ends. For each selection, colchicine treatments were applied separately. The experiment was designed to examine four colchicine concentrations including a negative control and four exposure periods. Five replications were performed for each treatment with each replication consisting of 10 stem pieces from an individual seedling per petri dish. Fig. 2. Average number of shoots produced through indirect organogenesis from colchicine treatments of pummelo selections 5-1-99-3, cybrid Hirado Buntan (CHBP), HBJL-5, and HBJL-7. Mean separations at each exposure time was obtained by calculating SEM at 5% level. Fig. 1. (A) Indirect organogenesis from callus produced at the cut ends of the stem pieces placed on DBA-3 shoot induction medium. (B) Browning of seedling stem-piece explants caused by higher colchicine concentration and duration. (C) Top-worked tetraploid tree derived from the cybrid Hirado Buntan (CHBP) selection on C-35 citrange rootstock. (D) Top-worked tetraploid tree derived from the 5-1-99-3 selection on C-35 citrange. HORTSCIENCE VOL. 49(7) JULY 2014 945 PROPAGATION AND TISSUE CULTURE

Production of Colchicine-induced Autotetraploids in Pummelo (Citrus grandis Osbeck) through Indirect Organogenesis