‘Garbí’ Mandarin: A New Late-maturing Triploid Hybrid

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Pablo Aleza Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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José Cuenca Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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José Juárez Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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José A. Pina Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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Luis Navarro Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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Citrus is the most extensively produced fruit tree crop in the world (FAO, Food and Agriculture Organization, 2009). There are two clearly differentiated markets: fresh fruit and processed juice. In 2007, the main citrus fruit-producing countries were China (17.9%), Brazil (17.8%), the Mediterranean countries (17.1%), and the United States (8.5%) (FAO, Food and Agriculture Organization, 2009). These areas account for more of two-thirds of the total production of citrus fruits. In the Mediterranean area, citrus fruits are primarily produced for the fresh fruit market. Spain is the principal producer in the area with a total planted area of 330,000 ha and a production ≈6.3 million tons.

Seedlessness is one of the most important characteristics for mandarin on the fresh fruit market, because consumers do not accept seeded fruits. Parthenocarpy is an essential trait for seedless fruit production, and this characteristic is present in citrus germplasm. Triploidy gives rise to seedless commercial cultivars. However, triploid plants have very low fertility, are generally sterile, and do not induce seeds in other cultivars by cross-pollination (Frost and Soost, 1968). Several methods have been developed to obtain triploid citrus (Navarro et al., 2002; Ollitrault et al., 2008). One exploits natural events of polyploidization such as 2n gametes using embryo rescue and flow cytometry to select triploids in 2x × 2x crosses. Second meiotic division restitution has been proposed for diploid megagametophyte development in clementine (Luro et al., 2004), whereas Chen et al. (2008) proposed first meiotic division restitution in sweet oranges.

In Spain, the structure of mandarin cultivars poses several problems. Mandarins include satsumas, clementines, and mandarin hybrids. Satsumas are harvested from the beginning of September until the beginning of November. They are cultivars with a high degree of parthenocarpy and have sterile pollen and ovules. Clementines are the most representative cultivars of mandarin in Spain. They are picked from mid-September until mid-February. They are self-incompatible, but pollen and ovules are viable, being able to pollinate and to be pollinated by other cultivars. Mandarin hybrids, like ‘Nova’ [C. clementina × (C. paradisi × C. tangerina)], ‘Fortune’ (C. clementina × C. tangerina), and ‘Ortanique’ tangor (natural hybrid between mandarin and C. sinensis), were introduced in the Spanish citriculture to cover the demand of late-season mandarins by international markets. They enable the harvesting period to be extended until May. These hybrids are also self-incompatible, but pollen and ovules are viable and cross-pollinate with clementines, producing fruits with seeds in both groups of cultivars.

A triploid breeding program has been carried out in Spain since 1996 based on sexual hybridization, embryo rescue, and ploidy analysis by flow cytometry (Navarro et al., 2002). The objective is to produce new high–quality, late-season and seedless triploid mandarin hybrids. In this article, we describe ‘Garbí’ mandarin, a new triploid hybrid developed within this program characterized by its high-quality, late-season ripening and seedless fruits.

Origin

‘Garbí’ is a new triploid hybrid obtained from a cross between diploid ‘Fortune’ mandarin and diploid tangor ‘Murcott’ (C. reticulata × C. sinensis). Anthers of ‘Murcott’ tangor were removed from flowers collected in preanthesis and dried in petri dishes over silica gel in a desiccator. Dried dehisced anthers were stored in small petri dishes at –20 °C. Controlled cross-pollination was conducted by applying one anther from the paternal parent to receptive stigma of ‘Fortune’ mandarin flowers in Spring 1996. Approximately 100 flowers of ‘Fortune’ mandarin were pollinated and 45 fruits were collected, which contained 129 small seeds produced by 2x × 2x crosses. One hundred twenty-two embryos were isolated from these seeds and cultured in vitro in the culture medium described by Murashige and Skoog (MS) (1962) with 50 g·L−1 sucrose, 500 mg·L−1 malt extract supplemented with vitamins (100 mg·L−1 i-inositol, 1 mg·L−1 pyridoxine hydrochloride, 1 mg·L−1 nicotinic acid, 0.2 mg·L−1 thiamine hydrochloride, 4 mg·L−1 glycine) and 8 g·L−1 Bacto agar (MS culture media). After germination, 116 plantlets were subcultured in an elongation medium, which consisted of the MS culture media without vitamins. Cultures were maintained at 24 ± 1 °C, 60% humidity, and 16-h daily exposure to 40 μE·m−2·s−1 illumination. Ploidy level of all plants was analyzed by flow cytometry in the flow cytometer Ploidy Analyzer (Partec®; Partec GmbH, Münster, Germany). Small pieces of leaves, measuring ≈0.5 cm2, were taken from the in vitro-growing plants and chopped together with a piece of leaf from a control diploid plant, placed in a Partec® CyStain ultraviolet Precise P nuclei extraction buffer, stained with DAPI, and analyzed in the cytometer. All the plantlets were triploid and were grafted onto ‘Carrizo’ citrange rootstock (C. sinensis × P. trifoliata) for field evaluation at IVIA (Instituto Valenciano de Investigaciones Agrarias, located in Moncada, Valencia, Spain) in 1998. ‘Garbí’ mandarin flowered for the first time in Spring 2002 and in 2004 it was selected because of its high fruit quality and propagated in a second evaluation plot to confirm its uniformity and stability. Ploidy level of ‘Garbí’ mandarin was confirmed by cytology using the hematoxylin staining technique of Sass (1958) and modified by Tusa et al. (1990).

Description

Description has been done with data taken from five trees growing at IVIA plots essentially following the Guidelines for the Conduct of tests for distinctness, uniformity, and stability for Citrus L. Group 1 mandarins from the International Union for the Protection of New Varieties of Plants (UPOV, International Union for the Protection of New Varieties of Plants, 2009).

The ‘Garbí’ mandarin tree is vigorous, obloid in shape, and exhibits drooping growth. Main branches have thorns of ≈30 mm in length, although on new branches ≈80% of the nodes have thorns measuring an average of 7.2 mm long.

‘Garbí’ mandarin fruits are seedless in an open-pollinated environment. They reach the optimum maturity at the end of March when the ratio solids/acids of the fruits is close to nine, although they can be harvested from mid-February to the end of April. Fruit characteristics are described in Table 1. Fruits are easy to peel like ‘Nova’ mandarin, obloid in shape, with a diameter slightly over 60 mm; the broadest part is central, circular shape in transverse section, absent neck; and the general shape of the proximal part is slightly rounded and very homogeneous (Fig. 1). Fruits do not have areola or persistent style nor do they have radial grooves at the distal end. Fruit rind is medium orange in color (Citrus Color Index = 13) (Jiménez-Cuesta et al., 1981) similar to ‘Clemenules’ clementine (C. clementina Hort. Ex Tan.) with reduced thickness (average 2.5 cm). The albedo is white and the flesh is orange in color. Fruits have absent or weak rudimentary segments and a medium number of well-developed segments (10 to 11 segments per fruit) and lack a navel. At maturity, the fruits have high total soluble sugars and acidity content, ≈2% of acid concentration, and 17 °Brix. Solids/acids ratio increased through the season (Fig. 2), primarily because of the increase in solids. If we compare the ratio of ‘Garbí’ mandarin with its parents at the optimum ripening stage (second half of March), the ratio of ‘Garbí’ mandarin (8.9) is lower than ‘Fortune’ mandarin (9.5) and ‘Murcott’ tangor (10.3) mainly as a result of a major quantity in acids of ‘Garbí’ mandarin. The flavor is slightly acidic like ‘Fortune’ mandarin with strong strength of fiber, easy-to-eat texture of the segments, and pleasant aroma, resembling ‘Murcott’ tangor.

Table 1.

Summary of fruit quality characteristics of ‘Garbí’mandarin on ‘Carrizo’ citrange rootstock.

Table 1.
Fig. 1.
Fig. 1.

Fruit of triploid ‘Garbí’ mandarin.

Citation: HortScience horts 45, 1; 10.21273/HORTSCI.45.1.139

Fig. 2.
Fig. 2.

Comparison of the ratio solids/acids evolution between ‘Garbí’ mandarin and its parents. Data are the average of three seasons and the bars represent the sd.

Citation: HortScience horts 45, 1; 10.21273/HORTSCI.45.1.139

The leaves of ‘Garbí’ mandarin are evergreen, simple with a long leaf blade (average length 11.5 cm) and medium width of the leaf blade (average 3.8 cm). The margin of the leaves has crenate incisions and an acute shape of the apex. The petiole is medium in length (average 13.6 mm) with small wings. Leaves are very characteristic and different from other mandarin cultivars.

‘Garbí’ mandarin flowers are hermaphrodite and white. Flowering occurs in April and many borne flowers are produced. Pollen fertility is very low with only 0.4% of pollen grains of ‘Garbí’ mandarin germinating in vitro culture as compared with ‘Fortune’ mandarin pollen grains of which over 82% germinated.

Controlled cross-pollinations have been carried out among ‘Garbí’ mandarin, ‘Loretina’ clementine, and ‘Fortune’ mandarin. Fifty flowers of ‘Loretina’ clementine with receptive stigma were pollinated by applying one anther from ‘Garbí’ mandarin and seedless fruits were obtained. Also, 50 flowers of ‘Garbí’ mandarin were pollinated with pollen from ‘Fortune’ mandarin and an average of 0.3 seeds were obtained per fruit. In open pollination, seeded fruits of ‘Garbí’ mandarin were very rare. These results confirm that this new triploid cultivar is essentially seedless and that seed formation in clementines is not induced by cross-pollinization.

Trees of ‘Garbí’ mandarin and several mandarin varieties are planted in experimental plots at IVIA that have a high level of Alternaria alternata inoculum. ‘Fortune’ mandarin and ‘Minneola’ tangelo (C. paradisi × C. tangerina) trees display severe A. alternata symptoms in leaves and fruits, and ‘Nova’ mandarin trees display mild to medium symptoms, whereas no symptoms have ever been observed in leaves or fruits of ‘Garbí’ mandarin.

‘Garbí’ mandarin was analyzed with 14 simple sequence repeat (SSR) markers by capillary electrophoresis. The size of alleles in nucleotides for each SSR marker is described in Table 2 (Froelicher et al., 2008; Kijas et al., 1997; Luro et al., 2008) and these results clearly distinguish ‘Garbí’ mandarin from other cultivated commercial cultivars on an international level. Genetic analysis with the microsatellite loci Ci05A05, Ci06B07, TAA 41, and Mest 15 indicated that origin of ‘Garbí’ mandarin was 2n gamete of ‘Fortune’ mandarin as a result of the presence of two alleles of this cultivar and one allele of ‘Murcott’ tangor. Also, molecular markers will help protect breeders’ rights and to control traceability of nursery-propagated plants.

Table 2.

Genetic analysis with simple sequence repeat (SSR) markers of ‘Garbí’ mandarin and different cultivars of mandarin.z

Table 2.

Availability

Protection of Plant Breeders’ Rights of the cultivar has been requested in the European Union, Morocco, Egypt, South Africa, and Turkey and also a U.S. Plant Patent has been requested. IVIA holds the rights to the cultivar and the Fundación de la Comunidad Valenciana para la Investigación Agroalimentaria (AGROALIMED) is handling these rights for commercial propagation under royalty agreements with licensed nurseries. Pathogen-free plants of ‘Garbí’ mandarin have been obtained by shoot-tip grafting in vitro according to the methodology described by Navarro et al. (1975) and healthy budwood has been released to 22 Spanish nurseries that have already signed propagation agreements to produce certified plants.

Literature Cited

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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Luro, F. , Costantino, G. , Terol, J.F. , Argout, X. , Allario, T. , Wincker, P. , Talon, M. , Ollitrault, P. & Morillon, R. 2008 Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other citrus species and their effectiveness for genetic mapping BMC Genomics 9 1 13

    • Search Google Scholar
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  • Luro, F. , Maddy, F. , Jacquemond, C. , Froelicher, Y. , Morillon, R. , Rist, D. & Ollitrault, P. 2004 Identification and evaluation of diplogyny in clementine (Citrus clementina) for use in breeding. En XI Eucarpia Symposium on Fruit Breeding and Genetics Acta Hort. 663 841 847

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 15 473 479

  • Navarro, L. , Juárez, J. , Aleza, P. & Pina, J.A. 2002 Recovery of triploid seedless mandarin hybrids from 2n × 2n and 2n × 4n crosses by embryo rescue and flow cytometry Proc. of the 10th IAPTC&B Congress, Plant Biotechnology 2002 and Beyond Orlando, FL 541 544

    • Search Google Scholar
    • Export Citation
  • Navarro, L. , Roistacher, C.N. & Murashige, T. 1975 Improvement of shoot-tip grafting in vitro for virus-free citrus J. Amer. Soc. Hort. Sci. 100 471 479

    • Search Google Scholar
    • Export Citation
  • Ollitrault, P. , Dambier, D. , Luro, F. & Froelicher, Y. 2008 Ploidy manipulation for breeding seedless triploid citrus Plant Breed. Rev. 20 323 354

    • Search Google Scholar
    • Export Citation
  • Sass, J.E. 1958 Botanical microtechnique Iowa State Univ. Press Ames, IA 73 75

  • Tusa, N. , Grosser, J.W. & Gmitter, F.G. 1990 Plant regeneration of ‘Valencia’ sweet orange, ‘Femminello’ lemon and the interspecific somatic hybrid following protoplast fusion J. Amer. Soc. Hort. Sci. 115 1043 1046

    • Search Google Scholar
    • Export Citation
  • UPOV, International Union for the Protection of New Varieties of Plants 2009 20 July 2009 <http://www.upov.int/en/publications/tg-rom/tg201/tg_201_1.pdf>.

  • Fruit of triploid ‘Garbí’ mandarin.

  • Comparison of the ratio solids/acids evolution between ‘Garbí’ mandarin and its parents. Data are the average of three seasons and the bars represent the sd.

  • Chen, C. , Lyon, M.T. , O'Malley, D. , Federici, C.T. , Gmitter, J. , Grosser, J.W. , Chaparro, J.X. , Roose, M.L. & Gmitter, F.G. 2008 Origin and frequency of 2n gametes in Citrus sinensis × Poncirus trifoliata and their reciprocal crosses Plant Sci. 174 1 8

    • Search Google Scholar
    • Export Citation
  • FAO, Food and Agriculture Organization 2009 20 July 2009 <http://faostat.fao.org/site/567/default.aspx#ancor>.

  • Froelicher, Y. , Dambier, D. , Bassene, J.B. , Costantino, G. , Lotfy, S. , Didout, C. , Beaumont, V. , Brottier, P. , Risterucci, A.M. , Luro, F. & Ollitrault, P. 2008 Characterization of microsatellite markers in mandarin orange (Citrus reticulata Blanco) Molecular Ecology Resources 8 119 122

    • Search Google Scholar
    • Export Citation
  • Frost, H.B. & Soost, R.K. 1968 Seed reproduction, development of gametes and embryos 290 324 Reuther W. , Batchelor L.D. & Webber H.B. The citrus industry Vol. 2 University of California Berkeley, CA

    • Search Google Scholar
    • Export Citation
  • Jiménez-Cuesta, M. , Cuquerella, J. & Martínez-Jávega, J.M. 1981 Determination of a color index for citrus fruit degreening En Proceedings of 4th International Citrus Congress International Society of Citriculture Tokyo, Japan 2 750 752

    • Search Google Scholar
    • Export Citation
  • Kijas, J.M.H. , Thomas, M.R. , Fowler, J.C.S. & Roose, M.L. 1997 Integration of trinucleotide microsatellites into a linkage map of citrus Theor. Appl. Genet. 94 701 706

    • Search Google Scholar
    • Export Citation
  • Luro, F. , Costantino, G. , Terol, J.F. , Argout, X. , Allario, T. , Wincker, P. , Talon, M. , Ollitrault, P. & Morillon, R. 2008 Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other citrus species and their effectiveness for genetic mapping BMC Genomics 9 1 13

    • Search Google Scholar
    • Export Citation
  • Luro, F. , Maddy, F. , Jacquemond, C. , Froelicher, Y. , Morillon, R. , Rist, D. & Ollitrault, P. 2004 Identification and evaluation of diplogyny in clementine (Citrus clementina) for use in breeding. En XI Eucarpia Symposium on Fruit Breeding and Genetics Acta Hort. 663 841 847

    • Search Google Scholar
    • Export Citation
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol. Plant. 15 473 479

  • Navarro, L. , Juárez, J. , Aleza, P. & Pina, J.A. 2002 Recovery of triploid seedless mandarin hybrids from 2n × 2n and 2n × 4n crosses by embryo rescue and flow cytometry Proc. of the 10th IAPTC&B Congress, Plant Biotechnology 2002 and Beyond Orlando, FL 541 544

    • Search Google Scholar
    • Export Citation
  • Navarro, L. , Roistacher, C.N. & Murashige, T. 1975 Improvement of shoot-tip grafting in vitro for virus-free citrus J. Amer. Soc. Hort. Sci. 100 471 479

    • Search Google Scholar
    • Export Citation
  • Ollitrault, P. , Dambier, D. , Luro, F. & Froelicher, Y. 2008 Ploidy manipulation for breeding seedless triploid citrus Plant Breed. Rev. 20 323 354

    • Search Google Scholar
    • Export Citation
  • Sass, J.E. 1958 Botanical microtechnique Iowa State Univ. Press Ames, IA 73 75

  • Tusa, N. , Grosser, J.W. & Gmitter, F.G. 1990 Plant regeneration of ‘Valencia’ sweet orange, ‘Femminello’ lemon and the interspecific somatic hybrid following protoplast fusion J. Amer. Soc. Hort. Sci. 115 1043 1046

    • Search Google Scholar
    • Export Citation
  • UPOV, International Union for the Protection of New Varieties of Plants 2009 20 July 2009 <http://www.upov.int/en/publications/tg-rom/tg201/tg_201_1.pdf>.

Pablo Aleza Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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José Cuenca Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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José Juárez Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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José A. Pina Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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Luis Navarro Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias. Ctra. Moncada a Náquera Km. 4.5, Apartado Oficial 46113, Moncada, Valencia, Spain

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Contributor Notes

This work is jointly financed by the AGL2008-00596-MCI and Prometeo 2008/121 Generalitat Valenciana projects.

To whom reprint requests should be requested; e-mail lnavarro@ivia.es.

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  • Fruit of triploid ‘Garbí’ mandarin.

  • Comparison of the ratio solids/acids evolution between ‘Garbí’ mandarin and its parents. Data are the average of three seasons and the bars represent the sd.

 

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