Implementation of extensive citrus triploid breeding programs based ...

Tree Genetics & Genomes (2012) 8:1293–1306 DOI 10.1007/s11295-012-0515-6

ORIGINAL PAPER

Implementation of extensive citrus triploid breeding programs based on 4x×2x sexual hybridisations P. Aleza & J. Juárez & M. Hernández & P. Ollitrault & L. Navarro

Received: 3 August 2011 / Revised: 30 January 2012 / Accepted: 3 May 2012 / Published online: 19 May 2012 # Springer-Verlag 2012

Abstract Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market, and mandarin triploid hybrids have this trait. Triploid citrus plants can be recovered by 4x×2x hybridisations using non-apomictic genotypes as female parents. In this study, we characterise the type of seeds obtained in 4x×2x hybridisations and the ploidy level of plants recovered from each type of seed. The majority of the plants recover were triploid (98.3 %), but a few diploid, tetraploid and pentaploid plants were also produced and their genetic origin was analysed by simple sequence repeat (SSR) markers. We also analysed the influence of parents and environmental conditions on the efficiency of recovery triploid hybrids. In this work, we present an effective methodology to recover triploid hybrids from 4x×2x hybridisations based on in vitro embryo rescue and determination of ploidy level by flow cytometry that allow us to recover more than 4,400 triploid hybrids from more than 60 parental combinations. Keywords Tetraploid . Pentaploid . Mandarin . Embryo rescue . Flow cytometry . SSR markers Communicated by A. Dandekar P. Aleza : J. Juárez : M. Hernández : P. Ollitrault (*) : L. Navarro (*) Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Ctra. Moncada-Náquera km 4.5, 46113 Moncada, Valencia, Spain e-mail: [email protected] e-mail: [email protected] P. Ollitrault TGU AGAP, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Avenue Agropolis—TA A-75/02, 34398 Montpellier cedex 5, France

Introduction Seedlessness is one of the most important characteristics for mandarins on the fresh fruit market, since consumers do not accept seedy fruits. The creation of triploid hybrids is an important breeding strategy to develop new commercial varieties of seedless citrus (Ollitrault et al. 2008). Triploid plants are generally considered an evolutionary dead-end, since they generally give rise to aneuploid gametes with very low fertility (Otto and Whitton 2000). Predominantly trivalent associations and also a high number of bivalent and univalent associations are formed during meiosis in citrus triploid hybrids (Cameron and Frost 1968). Moreover, abortion of megasporogenesis during the period between the embryo-sac first divisions and the fecundated egg cell is common (Fatta del Bosco et al. 1992). For these reasons, citrus triploid hybrids are generally sterile, although they can occasionally produce fruits with very few seeds and induce seed formation in the fruit of other cultivars by cross-pollination. Triploid citrus plants can be recovered directly from crosses between two diploid genotypes resulting from the union of a 2n megagametophyte with a haploid pollen (Esen and Soost 1971a, 1973; Geraci et al. 1975; Cuenca et al. 2011) or by hybridisation between diploid and tetraploid parents (Esen and Soost 1971b; Esen et al. 1978; Cameron and Burnett 1978). In 2x×2x hybridisations, the frequency of 2n gametes is generally low (Cameron and Frost 1968; Esen and Soost 1971a; Geraci et al. 1975), and extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation of large progenies (Aleza et al. 2010). Citrus breeding is hampered by apomixis of most genotypes both at diploid and tetraploid level. In Citrus, apomixis is determined by adventitious embryony from nucellar

1294

cells (Koltunow 1993). The majority of citrus genotypes are apomictic, with the exception of citrons (Citrus medica L.), pummelos (Citrus maxima (L.) Osb), clementines (Citrus clementina Hort. ex Tan.) and some mandarin hybrids. In seeds of apomictic citrus genotypes, formation of the nucellar embryos can be initiated before fertilisation (Wakana and Uemoto 1987), and competition between the zygotic and nucellar embryos generally results in the failure of the development of the zygotic embryo (Frost and Soost 1968; Koltunow 1993). This characteristic is a strong limitation for using apomictic tetraploid genotypes as female parents in 4x×2x hybridisations. Aleza et al. (2009b) developed an efficient method of obtaining stable tetraploid plants of non-apomictic citrus genotypes by combining shoot-tip grafting in vitro (Navarro and Juárez 2007) and treatment of shoot-tips with colchicine and oryzalin. This technique applied to adult material, allows avoidance of the long juvenile phase associated with tetraploid recovery from nucellar seedlings (Aleza et al. 2011), and these plants can be used as female parents in 4x×2x hybridisations. The 4x×2x hybridisations have been little used in the recovery of citrus triploid hybrids due to the difficulty of producing tetraploid plants from non-apomictic genotypes; consequently, there is few information available about this approach. Frost (1943) reported that a tetraploid grapefruit (Citrus paradisi Macf.) and a tetraploid ‘Lisbon’ lemon (Citrus limon (L.) Burm f.) produced triploids in crosses with diploid pollen parents. Later, Cameron and Frost (1968), Esen et al. (1978) and Cameron and Burnett (1978) performed different 4x×2x hybridisations. Plant regeneration was done without the help of in vitro techniques and ploidy level analysis was conducted using cytogenetic methods. Grosser and Gmitter (2011) obtained few nonapomictic allotetraploid somatic hybrids that have been used as female parents in interploid crosses to generate grapefruitpummelo triploid hybrids. Implementation of extensive and efficient triploid breeding programs based on 4x×2x hybridisations requires the availability of non-apomictic tetraploid genotypes to be used as female parents, the utilisation of in vitro culture techniques to improve germination rates (Aleza et al. 2010), flow cytometry-based techniques for ploidy level analysis (Ollitrault and Michaux-Ferriere 1992; Ollitrault et al. 1996; Navarro et al. 2002a) and knowledge about the type of seeds recovered, the ploidy level of plants produced and their genetic origin. In this work, we have studied the main factors affecting recovery of citrus triploid plants using 4x×2x hybridisations. We have characterised the different types of seeds obtained from 4x×2x hybridisations. We have analysed different factors influencing the behaviour of embryos cultured in vitro, the ploidy level of plants regenerated from

Tree Genetics & Genomes (2012) 8:1293–1306

each seed type, and how parents influence triploid hybrid recovery using this technology. These studies enabled us to establish an optimised process for an extensive citrus triploid breeding program. Our breeding program has given rise to over 4,400 triploid hybrids that are now being evaluated under field conditions. In addition, we have used simple sequence repeat (SSR) markers to analyse the genetic origin of diploid, triploid, tetraploid and pentaploid plants obtained in 4x×2x hybridisations.

Materials and methods Plant material All the genotypes used (Table 1) were from the Citrus Germplasm Bank of pathogen-free plants at the Instituto Valenciano de Investigaciones Agrarias (IVIA, Moncada, Spain; Navarro et al. 2002b). The two tetraploid clementine genotypes used as female parents are nonapomictic and were obtained by in vitro micrografting of shoot-tips, combined with treatment of shoot-tips with colchicine or oryzalin (Aleza et al. 2009b). For tetraploid ‘Clemenules’ clementine, 822 pollinations were done with ‘Imperial’, ‘Kara’, ‘Kinnow’, ‘Moncada’, ‘Ponkan’, ‘Primosole’ and ‘Willow leaf’ mandarins’, ‘Murcott’ and ‘Nadorcott’ tangors, ‘Seedy’ navel (SN) sweet orange and ‘Pink’ pummelo diploid male parents. For tetraploid ‘Fina’ clementine, 557 pollinations were done with ‘Fairchild’, ‘Kara’, ‘Kinnow’, ‘N-27’, ‘N’-15′, ‘Ñ’-6′, ‘Page’, ‘Ponkan’, ‘Scarlet’ and ‘Temple’ mandarins, ‘Murcott’ tangor and ‘SN’ sweet orange diploid male parents. The hybridisations were done over a 6-year period (from 2003 to 2009) and are part of the breeding program carried out in our laboratory since 1996 (Navarro et al. 2005). Additionally, we made a hybridisation between tetraploid ‘Clemenules’ clementine as female parent with diploid ‘Moncada’ mandarin male parent to describe and characterise seeds obtained in 4x×2x hybridisations. Pollination, seed extraction and characterisation Pollinations were carried out in trees grown in a large screenhouses and in the field. Anthers of the male parents were removed from flowers collected at pre-anthesis stage and dried in Petri dishes over silica gel in a desiccator. Dried dehisced anthers were stored in small Petri dishes at −20 °C. Flowers were hand-pollinated. Fruits were collected when ripe, and seeds were extracted and surface sterilised with a sodium hypochlorite solution (0.5 % active chlorine for 10 min.). Seeds were classified by size and developmental stage. Size was evaluated by

Tree Genetics & Genomes (2012) 8:1293–1306 Table 1 Genotypes used in 4x×2x hybridisations Tetraploid female parents

Scientific name

‘Clemenules’ clementine ‘Fina’ clementine

C. clementina Hort. ex Tan

Diploid male parents ‘Primosole’ mandarin ‘Imperial’ mandarin ‘Ponkan’ mandarin ‘Scarlet’ mandarin ‘Kinnow’ mandarin ‘Kara’ mandarin ‘Moncada’ mandarin ‘N’-15′ mandarin ‘Ñ’-6′ mandarin ‘Page’ mandarin ‘Fairchild’ mandarin ‘Willow leaf’ mandarin ‘Temple’ mandarin ‘Seedy’ navel sweet orange ‘Pink’ pummel ‘Murcott’ tangor ‘Nadorcott’ tangor

dishes, making sure that seeds did not touch each other. Petri dishes were scanned at 150 pp in an Epson® Perfection 4870 Photo scanner. Images were analysed using Matrox® software, which gives automatic exact measurements of area of the seeds. Embryo rescue and plant recovery

C. unshiu x (C. deliciosa x ?) C. reticulata Blanco

C. nobilis x C. deliciosa C. unshiu x C. nobilis C. clementina x (C. unshiu x C. nobilis) (C. paradisi x C. tangerina) x C. clementina C. clementina x (C. paradisi x C. tangerina) C. deliciosa Ten. C. temple Hort. ex Y. Tan. C. sinensis (L.) Osb. C. maxima (L.) Osb. C. reticulata x C. sinensis

measuring the area (mm2), and developmental stage was evaluated using morphological parameters. Seeds were considered developed when they had a normal appearance and were totally filled out and without any malformation. Seeds were considered undeveloped when development was incomplete, they were not totally filled out and were wrinkled or with the outer integument split (Fig. 1). Seeds were washed, dried and uniformly distributed in 9 cm Petri Fig. 1 Different types of seeds obtained in hybridisation between tetraploid ‘Clemenules’ clementine and diploid ‘Moncada’ mandarin. a Developed small seeds. b Undeveloped seeds. c Developed seeds (normal seeds)

1295

An experiment was done with the objective to compare the efficiency of embryo rescue in vitro with seed germination in the greenhouse to recover seedlings from developed small seeds obtained from open pollinated tetraploid ‘Clemenules’ clementine. Embryos were isolated with the aid of a stereoscopic microscope and were cultured under aseptic conditions in Petri dishes containing the Murashige and Skoog (1962) culture medium, with 50 g/L sucrose, 0.5 g/L malt extract and supplemented with vitamins (100 mg/L myoinositol, 1 mg/L pyridoxine hydrochloride, 1 mg/L nicotinic acid, 0.2 mg/L thiamine hydrochloride, 4 mg/L glycine) and 8 g/L Bacto agar (MS culture media). After germination, plants were transferred to 25×150 mm test tubes, with the same culture medium but without malt extract. Cultures were maintained at 24±1 °C, 60 % humidity and 16 h daily exposure to 40 μE m−2 s−1 illumination. Germination of seeds was done in trays with 96 alveoli, with 1 seed per alveolus, on a substrate composed of a mixture of 6 parts of peat moss (black/blond, in a mix 1:1) and 1 part of perlite, in a temperature controlled greenhouse (18–25 °C). Seeds were planted immediately after extraction from fruit. Ploidy level analysis Ploidy level was determined by flow cytometry following the methodology described by Aleza et al. (2009b). Each sample consisted of a small piece of leaf (~0.5 mm 2)

1296

Tree Genetics & Genomes (2012) 8:1293–1306

collected from each test tube plant, with a similar leaf piece taken from a diploid control plant. Samples were chopped together using a razor blade in the presence of a nuclei isolation solution (High Resolution DNA Kit Type P, solution A; Partec®, Münster, Germany). Nuclei were filtered through a 30-μm nylon filter and stained with DAPI solution (4-6-diamine-2-phenylindol; High Resolution DNA Kit Type P, solution B; Partec®). Following a 5-min incubation period, stained samples were run in a Ploidy Analyzer (Partec®, PA) flow cytometer equipped with a HBO 100 W high-pressure mercury bulb and both KG1 and BG38 filter sets. Histograms were analysed using dpac v2.0 software (Partec®), which determines peak position, coefficients of variation (CV), and the relative peak index of the samples. Transfer to soil Plants were transferred to pots containing a steam-sterilised artificial soil mix suitable to grow citrus (40 % black peat, 29 % coconut fibre, 24 % washed sand and 7 % perlite). Pots were enclosed in polyethylene bags that were closed with rubber bands and placed in a shaded area in a temperature-controlled greenhouse at 18–25 °C. After 8 to 10 days, the bags were opened, and after another 8 to 10 days, the bags were removed and the plants grown under regular greenhouse conditions (Navarro and Juárez 2007).

(François Luro, personal communication). The other diploid, tetraploid, pentaploid and hexaploid plants obtained in this work died during transplanting. The extraction of genomic DNA was done according to Dellaporta and Hicks (1983) with slight modifications. PCR amplifications were performed using a Thermocycler ep gradient S (Eppendorf®) in 10 mL final volume containing 0.8 U of Taq DNA polymerase (Fermentas®), 2 ng/mL of citrus DNA, 0.2 mM of wellRED (Sigma®) dye-labelled forward primer, 0.2 mM of non dye-labelled reverse primer, 0.2 mM of each dNTP, 10× PCR buffer and 1.5 mM MgCl2. The PCR protocol was as follows: denaturation at 94 °C for 5 min followed by 40 repeats of 30 s at 94 °C, 1 min at 50 °C or 55 °C, 45 s at 72 °C; and a final elongation step of 4 min at 72 °C. Capillary electrophoresis was carried out using a CEQ™ 8000 Genetic Analysis System (Beckman Coulter Inc.). The GenomeLab™ GeXP v.10.0 genetic analysis software was used for data collection and analysis. PCR products were initially denatured at 90 °C for 2 min, injected at 2 kV for 30 s and subsequently separated at 6 kV for 35 min. Alleles were sized, based on a DNA size standard (400 bp). Allele dosage was calculated using the MAC-PR (microsatellite DNA allele counting-peak ratio) method (Esselink et al. 2004), validated in citrus by Cuenca et al. (2011).

Molecular characterisation

Results and discussion

Eight triploid plants randomly selected from the hybridisation between tetraploid ‘Clemenules’ clementine and ‘Pink’ pummelo, one diploid plant obtained from tetraploid ‘Fina’ clementine and ‘Temple’ mandarin, two tetraploid plants obtained from tetraploid ‘Fina’ clementine and ‘Scarlet’ mandarin and seven pentaploid plants (six recovered from tetraploid ‘Clemenules’ clementine and ‘Pink’ pummelo and one from tetraploid ‘Fina’ clementine and ‘Kara’ mandarin) were analysed with 14 SSR markers. The SSR markers used were: TAA15, TAA41 (Kijas et al. 1997), mCrCIR07B05 (Froelicher et al. 2008), mCrCIR01C06, mCrCIR02D09, mCrCIR02G02, mCrCIR04H06, mCrCIR07D06 (Cuenca et al. 2011), mCrCIR02D04b, mCrCIR07F11 (Kamiri et al. 2011), mCrCIR03D12a (Aleza et al. 2011) mest56, mest104 and mest192

Seed description and ploidy level of recovered plantlets

Table 2 Type and size of seeds obtained in a hybridisation between tetraploid ‘Clemenules’ clementine and diploid ‘Moncada’ mandarin. Number and ploidy level of plants recovered

Type of seeds

Developed seeds Developed small seeds Undeveloped seeds

No. of seeds

1 122 19

A hybridisation was done between tetraploid ‘Clemenules’ clementine and diploid ‘Moncada’ mandarin, to correlate seed morphology with embryo ploidy. One hundred and forty-two seeds were obtained from 50 fruits; 123 were developed and 19 were undeveloped (Fig. 1). In the developed seeds, we clearly identified 2 different types, 122 were small seeds and 1 was a normal seed (Fig. 1a, c). The area of the normal seed was 69 mm2, whereas the average area of small seeds was 40±9 mm2; thus between 29 % and 45 % smaller than the normal seed. The average area of undeveloped seeds was 27±17 mm2 (Table 2). The embryo contained in the normal seed was well developed and the regenerated plant was triploid. Embryos were

Average area (mm2)

69 40±9 27±17

No. of recovered plants

1 116 1

Ploidy level Triploid

Pentaploid

1 114 0

0 2 1

Tree Genetics & Genomes (2012) 8:1293–1306

1297

rescued from 122 small seeds and cultured in vitro. One hundred and sixteen plantlets were obtained, of which 114 were triploid and two pentaploid. Only one pentaploid plant was recovered after embryo culture from undeveloped seeds (Table 2). In 4x×2x hybridisations, three seed types are obtained: undeveloped seeds, developed seeds (normal seeds) and developed small seeds. Generally, undeveloped seeds did not contain embryos (1/19), whereas the other two types contained one embryo per seed. These observations in 4x ‘Clemenules’ clementine X 2x ‘Moncada’ mandarin have been confirmed by the others 22 parental combinations performed in this work (Tables 3 and 4). By the whole, 512 fruits were obtained from 1,379 pollinations and 2,524 developed seeds were recovered, of which 99.3 % (2,507/ 2,524) was of small size and only 0.7 % (17 / 2,524) was of normal size. Ninety-eight percent of the plants recovered from developed small seeds was triploid, whereas from normal seeds only 76 % was triploid. Although from normal seeds it is also possible to recover triploid plants, the efficiency is very low due to the very small number of normal seeds per fruit (on average 0.03 per fruit). Esen et al. (1978) conducted histological studies demonstrating that in 4x×2x hybridisations small seeds contained triploid embryos with pentaploid endosperms, and indicated that triploid embryos originate from reduced megagametophytes and haploid pollen. In addition, these authors proposed that the three to five ratio between the ploidy level of embryos and endosperm was responsible for seed size

reduction, since pentaploid endosperms grow more slowly and stop development prematurely. The same ploidy ratios were also obtained in small seeds containing triploid embryos produced in 2x×2x hybridisations (Esen and Soost 1971a). Factors affecting recovery of citrus triploid plants Comparison of embryo rescue in vitro and seed germination in greenhouse conditions With the objective to compare the efficiency of in vitro embryo rescue with seed germination in greenhouse from developed small seeds, 602 embryos were isolated and cultured in vitro and 384 developed small seeds were cultured under greenhouse conditions. The germination percentage of embryos rescued in vitro from small seeds was 97.5 % (587/602), whereas germination of small seeds in the greenhouse was 69.5 % (267/384). Therefore, embryo rescue significantly improved triploid hybrid production. Nevertheless, in 4x×2x hybridisations that produce a high number of small seeds, the relatively high germination percentage in the greenhouse can avoid the embryo rescue procedure, what it is impossible in 2x × 2x and 2x × 4x hybridisations (Starrantino and Recupero 1981; Navarro et al. 2002a; Aleza et al. 2010). In 4x×2x hybridisations using non-apomictic allotetraploid genotypes as female parents, Grosser and Gmitter (2011) recovered hundreds of triploid grapefruitpummelo hybrids directly from seeds without embryo

Table 3 Embryo rescue, plant regeneration, and ploidy level of plants obtained in 4x×2x hybridisations with ‘Clemenules’ clementine as female parent Tetraploid female parent

Diploid male parent

Clemenules Clemenules Clemenules Clemenules Clemenules Clemenules Clemenules Clemenules Clemenules Clemenules Clemenules

Nadorcotta Murcottb Karab Primosole Imperial Willow leaf Moncada Ponkan Kinnow SN Pink

a

No. of pollinated flowers

No. of fruits set

No. of small seeds

No. of embryos culture

No. of recovered plants

No. of diploid plants

No. of triploid plants

No. of triploid plants per fruit

No. of triploid plants in greenhouse

219 110 156 50 50 32 25 50 50 30 50 822

103 26 40 5 21 15 10 20 26 6 21 293

662 150 141 17 79 45 89 91 180 28 310 1,792

662 150 140 17 79 45 89 90 180 28 310 1,790

635 141 123 17 72 40 86 87 171 28 300 1,700

4 0 0 1 1 0 0 1 0 0 0 7

625 135 120 15 71 39 85 86 171 28 292 1,667

6.1 5.2 3.0 3.0 3.4 2.6 8.5 4.3 6.6 4.7 13.9 5.7±0.7c

450 115 116 15 70 23 64 61 143 24 292 1,373

Data are the average of 2 years

b

Data are the average of 3 years

c

95 % confidence interval

1298

Tree Genetics & Genomes (2012) 8:1293–1306

Table 4 Embryo rescue, plant regeneration and ploidy level of plants obtained in 4x×2x hybridisations with ‘Fina’ clementine as female parent Tetraploid female parent

Diploid male parent

No. of pollinated flowers

No. of fruits set

No. of small seeds

No. of cultured embryos

No. of obtained plants

No. of diploid plants

No. of triploid plants

Fina Fina Fina Fina Fina

Murcotta Page Ponkan Fairchild Kinnowa

52 50 25 50 50

15 21 9 22 22

52 70 13 98 67

51 70 13 98 67

49 65 13 92 62

0 0 0 0 0

49 65 13 92 62

Fina Fina Fina Fina Fina Fina Fina

Karaa N-27 N-15 Ñ-6 Scarlet Temple SN

80 25 50 25 50 50 50 557

10 8 32 8 24 32 16 219

34 14 177 19 50 99 22 715

34 14 177 19 50 99 22 714

30 13 169 19 50 96 21 679

0 0 0 0 0 1 0 1

28 12 166 19 50 95 21 672

a

Data are the average of 2 years

b

95 % confidence interval

rescue procedure. Therefore, production of triploid hybrids is easier in 4x×2x hybridisations than in the others triploid breeding methods. Fruit set, plant regeneration, ploidy level and transfer to soil The results of embryo rescue, plant regeneration, ploidy level and transfer to soil, of plants recovered in 4x×2x hybridisations, are shown in Tables 3 and 4. The data correspond to 1,379 pollinations conducted between 2003 and 2009. When tetraploid ‘Clemenules’ clementine was used as female parent, the fruit set averaged was 36 % (Table 3). The number of small seeds per fruit varied between 3.0 for ‘Willow leaf’ mandarin and 14.8 for ‘Pink’ pummelo. In total, 1,792 small seeds were obtained, and 1,790 of these contained 1 embryo per seed and the other 2 seeds were aborted. From the 1,790 embryos cultured in vitro, 1,700 plants were recovered. The average germination Fig. 2 Hybridisation between tetraploid ‘Clemenules’ clementine and diploid ‘Nadorcott’ tangor. a Embryos rescued from small seeds. b Germinated embryos after 1 month. c In vitro triploid plant obtained after 3 months of initial culture

No. of triploid plants per fruit 3.3 3.1 1.4 4.2 2.8 2.8 1.5 5.2 2.4 2.1 3.0 1.3 3.1±0.2b

No. of triploid plants in greenhouse

43 52 11 80 57 24 12 146 19 47 92 20 603

percentage was 95 % using the embryo rescue technique (Fig. 2a, b) fluctuating between 88 % for ‘Kara’ mandarin and 100 % for ‘Primosole’ mandarin and ‘SN’ sweet orange (Table 3). The recovery efficiency of citrus triploid plants was calculated as the number of triploid plants per harvested fruit. The average number of triploid plants obtained per fruit was 5.7±0.7 considering all the hybridisations done; this varied between 2.6 for ‘Willow leaf’ mandarin and 13.9 for ‘Pink’ pummelo (Table 3). From the 1,700 plants obtained, 1,667 were triploid (98.1 %; Fig. 2c), 7 were diploid (0.41 %), 8 were tetraploid (0.47 %), 9 were pentaploid (0.53 %), 5 were hexaploid (0.29 %) and 4 aneuploid (0.24 %; Fig. 3a–f). The general survival percentage in the transplant phase was 82.4 % and plants obtained were robust and vigorous. When tetraploid ‘Fina’ clementine was used as female parent, the fruit set average was 39 % (Table 4). The number of small seeds per fruit varied between 1.4

Tree Genetics & Genomes (2012) 8:1293–1306

1299

for ‘Ponkan’ mandarin and ‘SN’ sweet orange to 5.5 for ‘N’-15′ mandarin. In total, 715 small seeds were obtained and 714 of them contained 1 embryo per seed, and 1 seed was aborted. From the 714 embryos cultured in vitro, 679 plants were recovered. The average germination percentage was 95 % using the embryo rescue technique, fluctuating between 88 % for ‘Kara’ mandarin and 100 % for ‘Ponkan’, ‘Ñ’-6′ and ‘Scarlet’ mandarins (Table 4).

The average number of triploid plants obtained per fruit was 3.1±0.2 considering all hybridisations done; this varied between 1.3 for ‘SN’ sweet orange to 5.2 for ‘N’-15′ mandarin (Table 4). From the 679 plants obtained, 672 were triploid (99 %), 1 was diploid (0.15 %), 2 were pentaploid (0.29 %) and 4 aneuploid (0.59 %). The general survival percentage in the transplant phase was 89.7 % and the plants obtained were robust and vigorous.

Fig. 3 Flow cytometry analysis of hybrids ploidy in 4x×2x hybridizations. a Histogram of the control diploid plant (peak 1). b Histogram of the diploid control plant (peak 1) and triploid plant (peak 2). c Histogram of the diploid control plant (peak 1) and tetraploid plant

(peak 2). d Histogram of the diploid control plant (peak 1) and pentaploid plant (peak 2). e Histogram of the diploid control plant (peak 1) and hexaploid plant (peak 2). f Histogram of the diploid control plant (peak 1) and aneuploid plant (peak 2)

1300

Tree Genetics & Genomes (2012) 8:1293–1306

Fig. 4 Triploid plants obtained from 4x×2x hybridisations transplanted and cultivated in the greenhouse

In all the hybridisations done with the two tetraploid clementines, 17 normal seeds were obtained and 17 plantlets were recovered. Thirteen plantlets were triploid and four were tetraploid. Obtained plants had diploid (0.34 %), triploid (98.3 %), tetraploid (0.34 %), pentaploid (0.46 %), hexaploid (0.21 %) and aneuploid (0.34 %) ploidy levels. In 4x×2x hybridisations, the average number of triploid plants per fruit, considering both clementines, was 4.6. In 2x×2x hybridisations with the same clementines as female parents, the number of triploid plants per fruit averaged 0.19 (Aleza et al. 2010). In our breeding program based on 2x× 4x hybridisations, with the same clementines as female parents, the number of triploid hybrids per fruit averaged 2.7 (unpublished data). These results indicate clearly that 4x× 2x hybridisations are more effective in recovering citrus triploid hybrids than are 2x×2x or 2x×4x hybridisations.

To develop extensive triploid breeding programs based on sexual hybridisations, it is essential to master the transplant phase (Fig. 4). The survival percentage of transplanted triploid plants produced in 4x×2x hybridisations was over 84 % and was close to 90 % for the 2x×2x hybridisations (Aleza et al. 2010). In 2x×4x hybridisations, differences were observed in the transplant phase, depending on the type of seed that produced triploid plants, with higher success for normal seeds (93 %) than for undeveloped seeds (78 %; unpublished data). Cameron and Burnett (1978) found that tetraploid plants, obtained with colchicine treatments, used as female parents were a complicating factor due to variation in the ploidy level of their histogenic layers. Our work has clearly demonstrated that colchicine tetraploid plants obtained from shoot-tip treatments have great value as female parents in triploid breeding programs, and enable the production of large populations of triploid hybrids. During the past few years, new triploid genotypes have been obtained from 4x×2x hybridisations in the USA (Williams and Roose 2004), and some of these cultivars are now beginning to be commercially propagated. Effect of parents and environmental conditions on recovery efficiency of citrus triploid hybrids We have studied effect of parents and environmental conditions on triploid hybrids production in all 4x×2x sexual hybridisations done over 5 year’s period. Triploid hybrids were obtained more efficiently using ‘Clemenules’ clementine than using ‘Fina’ clementine; these gave 5.7±0.7 and 3.1±0.2

Table 5 Effect of male parent in recovery of citrus triploid plants by 4x×2x hybridisation Female parent

Male parent

Clemenules

Murcott

Clemenules

Kara

Clemenules

Nadorcott

Fina

Murcott

Fina

Kara

Fina

Kinnow

Year

No. of pollinate flowers

No. of fruit

No. of small seeds set

No. of cultured embryos

No. of obtained plants

No. of triploid plants

No. of triploid plants per fruit

2003

55

10

48

48

48

44

4.4

2005 2007 2005 2006 2007 2003 2007 2007 2008 2007 2009 2008 2009

25 30 51 75 30 139 80 27 25 30 50 25 25

14 2 10 21 9 85 18 7 8 6 4 9 13

92 10 51 70 20 609 53 21 31 22 12 27 40

92 10 51 70 19 609 53 21 30 22 12 27 40

84 9 42 63 18 583 52 19 30 19 11 27 35

82 9 41 61 18 573 52 19 30 18 10 27 35

5.9 4.5 4.1 2.9 2.0 6.7 2.9 2.7 3.8 3.0 2.5 3.0 2.7

Tree Genetics & Genomes (2012) 8:1293–1306

1301

Fig. 5 Triploid hybrids recovered per fruit over 5 years with tetraploid ‘Clemenules’ clementine as female parent and 11 different male parents

triploids per harvested fruit, respectively (Tables 3 and 4). We suggest that citrus triploid hybrid recovery is affected by the genotype of the female parent. However, these results have not been obtained from hybridisations done during the same year and with the same male parents. It is therefore important to estimate the variability associated with these two factors. Triploid production efficiency was estimated for tetraploid ‘Clemenules’ and ‘Fina’ clementines crossed with four

male parents over 6 years (Table 5). Differences were observed concerning the male parents. ‘Kara’ mandarin male parent gave the lowest number of triploid hybrids per fruit when crossed with the two tetraploid clementines, whereas the highest number of triploid hybrids per fruit was obtained using ‘Nadorcott’ tangor with ‘Clemenules’ clementine and ‘Murcott’ tangor with ‘Fina’ clementine. Bono et al. (2006) demonstrated that within the mandarin group, there are great

Fig. 6 Profiles obtained using the mest192 SSR marker in the tetraploid ‘Fina’ clementine, ‘Temple’ mandarin and polihaploid plant of tetraploid ‘Fina’

1302

differences in the number of seeds per fruit depending on the parents used for the crosses. To obtain a better estimation of environmental effects and their interactions with the male parent, we analysed the number of triploid plants recovered per fruit in all the crosses done with ‘Clemenules’ clementine over 5 years (Fig. 5). Variability was observed between years, for the same parental combinations. For example, in the cross with ‘Nadorcott’ tangor, the number of triploid hybrids per fruit varied between 2.9 in 2007 and 6.7 in 2003. Variability was also observed between hybridisations performed in the same year with different male parents. For example, for the 2006 hybridisations, the number of triploid hybrids per fruit oscillated between 2.9 for hybridisations with ‘Kara’ mandarin and 13.9 for hybridisations with ‘Pink’ pummelo. Moreover, the rank of the different male parents was not conserved between years; possibly indicating an important interaction

Tree Genetics & Genomes (2012) 8:1293–1306

between the genotype of the male parent and environmental conditions. Our results over 5 years of hybridisation suggest an interaction between male parents and environmental conditions in triploid production of 4x×2x hybridisations. Luro et al. (2004) showed that environmental conditions affect production of triploid hybrids in 2x×2x hybridisations, and Viloria and Grosser (2005) observed a temperature effect in citrus hybrid production efficiency based on 2x×4x hybridisations. Triploid hybrid production will be influenced by the ability of pollen to perform successful fecundation. This ability is dependent on the quality of pollen determined by the male genotype, but also environmental conditions, the compatibility level between female and male parents, and environmental effects on pollen tube germination. Studies of a few species have shown that environmental conditions affect pollen development (Young and Stanton 1990; Jóhannsson and

Fig. 7 Profiles obtained using the mCrCIR07F11 SSR marker in tetraploid ‘Clemenules’ clementine, ‘Scarlet’ mandarin and tetraploid plants with allele’s dosage

Tree Genetics & Genomes (2012) 8:1293–1306

Stephenson 1998), as well as pollen tube growth (Stephenson et al. 1992). One of the most important environmental factors that could affect pollen performance is the temperature during the pregamic phase. Indeed, temperature has been shown to affect pollen germination (Elgersma et al. 1989; Shivanna et al. 1991) and pollen tube kinetics within the style (Lewis 1942; Jefferies et al. 1982; Elgersma et al. 1989). It should be interesting to analyse the correlation between the pollen germination and the recovery of triploid hybrids by performing pollen viability and germination tests at the same time of pollinations. SSR analysis to determine the origin of various ploidy levels recovered from 4x×2x hybridisations To determine genetic origin of plants recovered from 4x×2x hybridisations, diploid, triploid, tetraploid and hexaploid plants were analysed with SSR markers. Eight triploid plants randomly selected from the hybridisation between tetraploid ‘Clemenules’ clementineבPink’ pummelo were analysed using the mCrCIR02D09 heterozygotic SSR marker for clementine with a different allele for ‘Pink’ pummelo. The results demonstrated that triploid plants are hybrids between both parents displaying two alleles from female parent and one from male parent.

1303

One diploid plant obtained from hybridisation between tetraploid ‘Fina’ clementine and ‘Temple’ mandarin was analysed using five heterozygotic SSR markers (TAA 41, mCrCIR01C06, mCrCIR07B05, mCrCIR07F11 and mest192), with one or two different alleles between parents. With all markers, the diploid plant displayed alleles corresponding to the female parent (Fig. 6) and we did not observe alleles specific to the male parent with any SSR markers. The ploidy level and genetic analysis allow us to conclude that the diploid plant was spontaneously obtained by a gynogenetic process from a reduced gamete of the tetraploid ‘Fina’ (n02x018). In citrus, haploid plants have been obtained by gynogenesis from diploid genotypes in 2x×2x (Esen and Soost 1971a; Toolapong et al. 1996) and 2x × 3x hybridisations (Oiyama and Kobayashi 1993; Germanà and Chiancone 2001), and after pollination with irradiated pollen (Froelicher et al. 2007; Aleza et al. 2009a). Two tetraploid plants obtained from the hybridisation between the tetraploid ‘Fina’ clementine and ‘Scarlet’ mandarin were analysed using ten heterozygotic SSR markers (TAA 41, mCrCIR07B05, mCrCIR01C06, mCrCIR02D09, mCrCIR02G02, mCrCIR07D06 mCrCIR02D04b, mCrCIR07F11, mCrCIR03D12a and mest104), with one or two different alleles between parents. In all cases, tetraploid plants showed specific clementine alleles with the

Fig. 8 Profiles obtained using mCrCIR07B05 SSR marker in tetraploid ‘Clemenules’ clementine, ‘Kara’ mandarin and pentaploid hybrid with allele’s dosage

1304

allele dosage following 1:1, 3:1 or 1:3 ratios (Fig. 7). This suggests that these two plants originated by autopollination (selfing). From 2,396 plants recovered, only 12 plants were tetraploid. This phenomenon therefore occurs at very low frequency (0.5 %) and it is in accordance with the fact that clementines are self-incompatible. Seven pentaploid plants recovered from 4x×2x hybridisations (six from hybridisation between tetraploid ‘Clemenules’

Tree Genetics & Genomes (2012) 8:1293–1306

clementine and ‘Pink’ pummelo, and one from tetraploid ‘Fina’ clementine and ‘Kara’ mandarin) were analysed using 12 SSR markers heterozygous for clementine, and displaying 1 or 2 different alleles between parents (TAA 41, mCrCIR07B05, mCrCIR01C06, mCrCIR02D09, mCrCIR02G02, mCrCIR07D06, mCrCIR04H06 mCrCIR02D04b, mCrCIR07F11, mCrCIR03D12a, mest56 and mest104). The results indicate that analysed plants were hybrids showing the allele specific to the male parent for each SSR marker (Fig. 8), and 2:2, 3:1 or 1:3 ratios between the clementine alleles. Ploidy level and genetic analysis of these pentaploid plants demonstrated that they have been produced by pollination of unreduced megagametophites (2n04x036) with haploid pollen (n0×09). Oiyama and Kobayashi (1991) also obtained pentaploid plants from 2x×2x hybridisations and concluded that they had arisen from the union of doubly unreduced female gametes with normal reduced male gametes. Pentaploid plants display a very weak aspect and poor growth compared with diploid, triploid and tetraploid plants.

Conclusions The different factors influencing the recovery of citrus triploid hybrids from 4x ×2x hybridisations were analysed. Triploid hybrids are found in developed seeds that are between 29 % and 45 % smaller than normal seeds, and generally the undeveloped seeds do not contain embryos. Recovery of triploid hybrids can be done by embryo rescue and also, although with lower efficiency, by direct germination of small seeds in the greenhouse. Female parent seem to affect triploid hybrid production and our results over 6 years of 4x×2x hybridisation suggest an interaction between male parents and environmental conditions in triploid production. Although the majority of the plants produced were triploids, a few diploid, tetraploid and pentaploid plants have been also obtained. We have demonstrated the genetic origin of these plants. In our laboratory, an extensive breeding program of citrus triploid hybrids, based on these hybridisation techniques, has been running since 2001. To date, we have obtained over 4,400 hybrids from more than 60 combinations with 38 different male parents. These are now being field evaluated to select new seedless varieties (Fig. 9).

Fig. 9 Triploid plants obtained from 4x×2x hybridisations growing in the field. a One-year-old triploid plants grafted onto C. macrophylla Wester. b Seven years old triploid plants grafted onto ‘Carrizo’ citrange (C. sinensis x P. trifoliata). c Ten years old triploid plants grafted onto ‘Carrizo’ citrange

Acknowledgements We thank J.M. Arregui, C. Ortega, A. Navarro, V. Ortega, and C. Martí for their technical assistance in the laboratory, and J.A. Pina, V. Lloris, J.M. Conchilla, F. Ahuir, D. Conchilla, A. Conchilla, R. López, and F.J. Martí for growing plants in the greenhouse and in the field. We also thank Dr. F. Luro from INRA (France) for providing unpublished SSRs markers. This work was supported by a grant (Prometeo/2008/121) from the Generalitat Valenciana, Spain and by a grant (AGL2008-00596) from the Ministry of Science and Innovation of Spain-Fondo Europeo de Desarrollo Regional (FEDER).

Tree Genetics & Genomes (2012) 8:1293–1306

References Aleza P, Juárez J, Hernández M, Pina JA, Ollitrault P, Navarro L (2009a) Recovery and characterization of a Citrus clementina Hort. ex Tan. ‘Clemenules’ haploid plant selected to establish the reference whole Citrus genome sequence. BMC Plant Biol 9:110 Aleza P, Juárez J, Ollitrault P, Navarro L (2009b) Production of tetraploid plants of non apomictic citrus genotypes. Plant Cell Rep 28:1837–1846 Aleza P, Juárez J, Cuenca J, Ollitrault P, Navarro L (2010) Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x×2x sexual hybridisation and its application to extensive breeding programs. Plant Cell Rep 29:1023–1034 Aleza P, Froelicher Y, Schwarz S, Hernández M, Juárez J, Morillon R, Navarro L, Ollitrault P (2011) Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependant of genotype and environment. Ann Bot 108:37–50 Bono R, Soler J, Buj A, Villalba D, Salvia J, Bellver R, Mares M, Méndez JV, Gavilá L (2006) El problema de la presencia de semillas en los frutos cítricos. Conselleria de Agricultura Pesca y Alimentación, Ficha Técnica serie citricultura Nº 5 Cameron JW, Burnett RH (1978) Use of sexual tetraploid seed parents for production of triploid citrus hybrids. HortSci 13:167–169 Cameron JW, Frost HB (1968) Genetic, breeding and nucellar embryony. In: Reuther W, Batchelor LD, Webber HJ (eds) The Citrus Industry, vol 1. University of California, Riverside, pp 325–370 Cuenca J, Froelicher Y, Aleza P, Juárez J, Navarro L, Ollitrault P (2011) Multilocus half tetrad analysis and centromere mapping in Citrus; evidences of SDR mechanism for 2n megagametophyte production and partial chromosome interference in mandarin cv Fortune. Heredity 107(5):462–470 Dellaporta J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Report 1:19–21 Elgersma A, Stephenson AG, Nijs APM (1989) Effects of genotype and temperature on pollen tube growth in perennial ryegrass (Lolium perenne L.). Sex Plant Reprod 2:225–230 Esen A, Soost RK (1971a) Unexpected triploids in citrus: their origin, identification and possible use. J Hered 62:329–333 Esen A, Soost RK (1971b) Tetraploid progenies from 2x–4x crosses of Citrus and their origin. J Am Soc Hortic Sci 97:410–414 Esen A, Soost RK (1973) Precocious development and germination of spontaneous triploid seeds in Citrus. J Hered 64:147–154 Esen A, Soost RK, Geraci G (1978) Seed set, size and development after 4x–2x and 4x–4x crosses in Citrus. Euphytica 27:283–293 Esselink GD, Nybom H, Vosman B (2004) Assignment of allelic configuration in polyploids using the MAC-PR (microsatellite DNA allele counting—peak ratios) method. Theor Appl Genet 109:402–408 Fatta Del Bosco S, Matranga G, Geraci G (1992) Micro and macrosporogenesis of two triploid hybrids of Citrus. In: Proceedings of 7th International Citrus Congress. International Society of Citriculture, Acireale, Italy. 1:122–124 Froelicher Y, Bassene JB, JedidI-Neji E, Morillon R, Bernardini G, Costantino G, Ollitrault P (2007) Induced parthenogenesis in mandarin: induction procedures and genetic analysis of plantlets. Plant Cell Rep 26(7):937–944 Froelicher Y, Dambier D, Bassene JB, Costantino G, Lotfy S, Didout C, Beaumont V, Brottier P, Risterucci AM, Luro F, Ollitrault P (2008) Characterization of microsatellite markers in mandarin orange (Citrus reticulata Blanco). Mol Ecol Resour 8(1):119–122 Frost HB (1943) Genetics and breeding. In: Webber HJ, Batchelor LD (eds) The Citrus Industry, vol I. University of California, Riverside, pp 817–913

1305 Frost HB, Soost RK (1968) Seed reproduction: development of gametes and embryos. In: Reuther W, Batchelor LD, Webber HB (eds) The Citrus Industry, vol 2. University of California, Barkley, USA, pp 290–324 Geraci G, Esen A, Soost RK (1975) Triploid progenies from 2x–2x crosses of Citrus cultivars. J Hered 66:177–178 Germanà MA, Chiancone B (2001) Gynogenetic haploids of Citrus after in vitro pollination with triploid pollen grains. Plant Cell Tissue Organ Cult 66:59–66 Grosser WJ, Gmitter FG (2011) Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus. Plant Cell Tissue Organ Cult 104:343–357 Jefferies CJ, Brain P, Stott KG, Belcher AR (1982) Experimental systems and mathematical model for studying temperature effects on pollen-tube growth and fertilization in plum. Plant Cell Environ 5:231–236 Jóhannsson MH, Stephenson AG (1998) Effects of temperature during microsporogenesis on pollen performance in Cucurbita pepo L. (Cucurbitaceae). Int J Plant Sci 159:616–626 Kamiri M, Stift M, Srairi I, Costantino G, El Moussadik A, Hmyene A, Bakry F, Ollitrault P, Froelicher Y (2011) Evidence for nondisomic inheritance in a Citrus interspecific tetraploid somatic between C. reticulata and C. lemon hybrid using SSR markers and cytogenetic analysis. Plant Cell Rep 30:1415–1425 Kijas JMH, Thomas MR, Fowler JCS, Roose ML (1997) Integration of trinucleotide microsatellites into a linkage map of Citrus. Theor Appl Genet 94:701–706 Koltunow AM (1993) Apomixis: embryo sacs and embryos formed without meiosis or fertilization in ovules. Plant Cell 5:1425–1437 Lewis D (1942) The physiology of incompatibility in plants. I. Effect of temperature. Proc R Soc London Ser B Biol Sci 131:13–26 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. In: XI Eucarpia Symposium on Fruit Breeding and Genetics. Acta Horticulturae 663:841–847 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 (2007) Shoot-tip grafting in vitro. In: Khan IA (ed) Citrus genetics, breeding and biotechnology. CABI Head Office, Wallingford, pp 353–364 Navarro L, Juárez J, Aleza P, Pina JA (2002a) Recovery of triploid seedless mandarin hybrids from 2n×2n and 2n×4n crosses by embryo rescue and flow cytometry. In: Proceedings of the 10th IAPTC&B Congress, Plant Biotechnology 2002 and Beyond. Orlando, Florida, USA, pp. 541–544 Navarro L, Pina JA, Juárez J, Ballester-Olmos JF, Arregui JM, Ortega C, Navarro A, Duran-Vila, N, Guerri J, Moreno P, Cambra M, Zaragoza S (2002b) The citrus variety improvement program in Spain in the period 1975–2001. In: Proceedings of the 15th Conf. Intern. Organization Citrus Virol., IOCV, Riverside, pp. 306–316 Navarro L, Juárez J, Aleza P, Pina JA, Olivares-Fuster O, Cuenca J, Julve JM (2005) Programa de obtención de híbridos triploides de mandarino en España. Phytoma 170:36–41 Oiyama I, Kobayashi S (1991) Citrus pentaploids from small seeds of diploid × diploid crosses. HortSci 26(3):292–293 Oiyama I, Kobayashi S (1993) Haploids obtained from diploid × triploid crosses of citrus. J Jpn Soc Hortic Sci 62:89–93 Ollitrault P, Michaux-Ferriere N (1992) Application of flow cytometry for citrus genetic and breeding. In: Proceeding of the 7th International Citrus Congress, International Society of Citriculture, Acireale, Italy. 1:193–198 Ollitrault P, Dambier D, Allent V, Luro F, Jacquemond C (1996) In vitro embryo rescue and selection of spontaneous triploid by flow cytometry for easy peeler citrus breeding. In: Proceeding of the

1306 8th Citrus International Congress. International Society of Citriculture. Sun City, South Africa. 1:254–258 Ollitrault P, Dambier D, Luro F, Froelicher Y (2008) Ploidy manipulation for breeding seedless triploid citrus. Plant Breed Rev 20:323–354 Otto SP, Whitton J (2000) Polyploid incidence and evolution. Annu Rev Genet 34:401–437 Shivanna KR, Linskens HF, Cresti M (1991) Response of tobacco pollen to high humidity and heat stress: viability and germinability in vitro and in vivo. Sex Plant Reprod 4:104–109 Starrantino A, Recupero G (1981) Citrus hybrids obtained in vitro from 2x females X 4x males. In: Proceedings 4th International Citrus Congress. International Society of Citriculture, Tokyo, Japan. 1:31–32 Stephenson AG, Lau TC, Quesada M, Winsor JA (1992) Factors that affect pollen performance. In: Wyatt R (ed) Ecology and evolution of plant reproduction. Chapman & Hall, New York, pp 119–134 Toolapong P, Komatsu H, Iwamasa M (1996) Triploids and haploid progenies derived from small seeds of ‘Banpeiyu’ pummelo,

Tree Genetics & Genomes (2012) 8:1293–1306 crossed with ‘Ruby Red’ grapefruit. J Jpn Soc Hortic Sci 65:255–260 Viloria Z, Grosser JW (2005) Acid citrus fruit improvement via interploid hybridization using allotetraploid somatic hybrid and autotetraploid breeding parents. J Am Soc Hortic Sci 130(3):392–402 Wakana A, Uemoto S (1987) Adventive embryogenesis in citrus I. The occurrence of adventive embryos without pollination or fertilization. Am J Bot 74:517–530 Williams TE, Roose ML (2004) ‘TDE2’ Mandarin hybrid (Shasta Gold® Mandarin), ‘TDE3’ Mandarin hybrid (Tahoe Gold® Mandarin) and ‘TDE4’ Mandarin hybrid (Yosemite Gold® Mandarin): Three New Mid and Late-Season Triploid Seedless Mandarin Hybrids from California. In: Proceedings of 10th International Citrus Congress. International Society of Citriculture. Agadir, Marruecos, 1:394–398 Young HJ, Stanton ML (1990) Influence of environmental quality on pollen competitive ability in wild radish. Science 248: 1631–1633