for ornamental use - Springer Link

Euphytica (2012) 184:389–399 DOI 10.1007/s10681-011-0607-7

Confirmation and characterization of interspecific hybrids of Passiflora L. (Passifloraceae) for ornamental use Eileen Azevedo Santos • Margarete Magalha˜es Souza • Priscilla Patrocıńio Abreu • Leo Duc Haa Carson Schwartzhaupt da Conceiçaõ • Iona´ Santos Arau´jo • Alexandre Pio Viana • Alex-Alan Furtado de Almeida • Joˆsie Cloviane de Oliveira Freitas

Received: 5 October 2009 / Accepted: 21 December 2011 / Published online: 31 December 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Three new varieties of Passiflora hybrids were developed from crosses between P. sublanceolata J. M. MacDougal (ex P. palmeri var. sublanceolata Killip) versus P. foetida var. foetida L. Twenty putative hybrids were analyzed. Hybridizations were confirmed by RAPD and SSR markers. The RAPD primer UBC11 (50 -CCGGCCTTAC-30 ) generated informative bands. The SSR primer A08FP1 amplified

E. A. Santos M. M. Souza (&) P. P. Abreu A.-A. F. de Almeida J. C. O. Freitas Departamento de Cieˆncias Biolo´gicas, Universidade Estadual de Santa Cruz (UESC), Pavilhaõ Jorge Amado, Rod. Ilheús-Itabuna, Km 16, Ilheús, BA 45662-000, Brazil e-mail: [email protected] L. D. H. C. S. da Conceiçaõ Empresa Brasileira de Pesquisa Agropecua´ria (Embrapa Cerrados), Rodovia Brası´lia-Fortaleza, BR020, Km 18 73310-970, Planaltina, DF, Caixa Postal 08223, Brazil I. S. Arau´jo Departamento de Cieˆncias Vegetais, Universidade Federal ´ rido (UFERSA), BR 110, Km 47, Pres. Rural do Semi-A Costa e Silva, Mossoro´, RN 59625-900, Brazil A. P. Viana Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Centro de Cieˆncias e Tecnologia Agropecua´ria, Av. Alberto Lamego, 2000, Parque Califo´rnia, Campos dos Goytacazes, RJ 28013-600, Brazil

species-specific fragments and heterozygote status was observed with the two parent bands 240 and 280 bp. The molecular markers generated by primers were analyzed in terms of the presence or absence of specific informative bands. The morphological characterization of the hybrids enabled their differentiation into three groups, identified as: (1) Passiflora ‘Alva’, composed of five hybrid plants with white flowers, large corona, light purple filaments at base, white and purple/white banding to apex; (2) P. ‘Aninha’, composed of six hybrid plants with pale pink flowers, corona filaments reddish/purple at base, white, purple/ white banding to apex; (3) P. ‘Priscilla’, composed of nine hybrid plants with white flowers, small corona, filaments dark purple at base, white and purple to apex. The genomic homology of parent plants was verified by cytogenetic analysis. Both parents were 2n = 22. Meiosis was regular in genitors and hybrids. Aneuploidy was observed at hybrid groups P. ‘Alva’ and P. ‘Priscilla’ (2n = 20). Other authors had already observed the same number of chromosomes for some P. foetida genotypes. Obtaining valuable interspecific hybrids opens up new perspectives to offer opportunities in agribusiness for producers and to arouse the interest of consumers into using passion flowers in the Brazilian ornamental plant market. Keywords Interspecific hybrid Passiflora ‘Alva’ Passiflora ‘Aninha’ Passiflora ‘Priscilla’ RAPD SSR

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Introduction Passion flowers are found in North and South America, the West Indies, the Galapagos Islands, Africa, Australia, the Philippines, and many islands in the Pacific Ocean, of which South America is home to 95% of all passion flowers (Vanderplank 2000). Thus it is believed that passion flowers originated in the Americas and were later spread to other regions of the world (Ulmer and MacDougal 2004; Vanderplank 2000). The Passifloraceae family has more than 700 species (Berry 2011). Until 2005, it had been estimated that the Passiflora genus comprised some 520 species (Cervi 2005). However, new species continue to be identified (Cervi and Linsingen 2010). In Brazil, most of the Passiflora species are found mainly in the center-north region (Ulmer and MacDougal 2004). Passiflora is protandrous hermaphrodite. Its anthers dehisce before the stigma becomes receptive, and the stigmas are receptive from flower opening to closing (Souza and Pereira 2011). The corona is composed of one to several series of white or colored filaments, placed around the androginophore and female sexual organs, a characteristic of the Passifloraceae family (Ulmer and MacDougal 2004). Most Passiflora species flower abundantly for a prolonged period, its flowers usually remaining open for 1 day only, except for some species such as P. bahiensis and P. eichleriana, whose flowers remain open for more than 24 h (Abreu et al. 2009). P. aurantia, P. cinnabarina and P. herbertiana stay open for up to three consecutive days (Ulmer and MacDougal 2004). Flower opening occurs mainly at daytime, such as with P. sublanceolata and P. foetida, but there are species whose flowers have nocturnal anthesis such as P. setacea and P. mucronata. In this genus, the species presenting sexual reproduction may involve both selfcompatible (P. foetida) and self-incompatible systems (P. sublanceolata); in this case, pollen produced in a particular flower cannot effectively fertilize other flowers produced on the same plant (Junqueira et al. 2005). Studies on the floral biology and pollination of various Passiflora species have reported the predominance of melittophily and the assistance of birds, but there are some species that are pollinated by bats (chiropterophily) (Benevides et al. 2009). Passiflora species have been used to decorate gardens and greenhouses in European countries since their introduction into the Old World, in the

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seventeenth century (Peixoto 2005; Ulmer and MacDougal 2004). Their inclusion in the ornamental plant market is especially due to the exuberance of their flowers, which display a range of colors and exotic forms, granting them peculiar beauty. In many tropical and subtropical countries of Latin America, despite the existing genetic diversity and a favorable climate for the cultivation of such species, the ornamental potential of passion flowers is not much explored, since they are not usually found in stores specializing in ornamental plants. Despite expansion of the use of Passiflora species for purely ornamental reasons, it is still restricted to a few species, like P. alata Dryand, P. cincinatta Mast and P. coccinea Aubl, that decorate hedges, walls and pergolas (Peixoto 2005; Abreu et al. 2009). Passiflora has also been used as an ornamental plant in the Northern Hemisphere (Peixoto 2005), where more than 400 hybrids have been produced and registered as of 2000 (Feuillet et al. 2000) for decoration purposes. In that respect, the commercialization of new hybrids may improve the ornamental plant market and provide business opportunities for small holdings (Abreu et al. 2009). In Latin American countries where Passiflora is a native species, it is not used for ornamental purposes. This is mainly due to the lack of improvement programs that focus on obtaining ornamental hybrids. Interspecific hybridization is a frequently used technique to transfer genes from disease-resistant genotypes to susceptible genotypes, and to produce ornamental hybrids. Sexual hybridization techniques are very simple (Bruckner and Otoni 1999; Ulmer and MacDougal 2004; Vanderplank 2000), and are habitually used in improvement programs that aim to obtain genotypes with characteristics that are intermediate to the genitors. Many Passiflora species can be crossed with no difficulties. Many interspecific hybrids have been obtained with Passiflora because incompatibility barriers are fragile (Meletti 2005). By 2003, about 570 hybrids for ornamental purposes were reported (Vanderplank et al. 2003), most of which present unique and varied sizes, colors and shapes. Passiflora foetida var. foetida was first described by Linnaeus (1753) and revised by Killip (1938). It presents interesting characteristics for indoors ornamentation due to its small size and abundant flowering. It is found in Puerto Rico, Jamaica and Antilles in addition to being widely distributed throughout South

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America, including Brazil. The species is characterized as herbaceous vine, stem cylindrical striatum. Leaves are pubescent on both sides and membranous; flowers are white/purple and the corona filaments are white with a purple/blue base. P. sublanceolata was revised by MacDougal (2004), and before that it was classified as P. palmeri var. sublanceolata (Killip 1938). It is endemic to the deserts of Baja California, and is distributed from Tabasco, in Mexico, to the Yucatan Peninsula. The species is characterized as climbing slender, densely hairy, with whole, serrated leaves, beautiful pink/red–purple flowers and small corona, white or white with pink, and pink or purple bands (Killip 1938; Vanderplank 2000; Ulmer and MacDougal 2004). In addition to this, such species bloom and yield fruits throughout the year, this also benefits their use as parents in genetic improvement programs. Several techniques may be used to identify hybrids, from the ones based on morphological characters, which are simpler and lower in cost, up to the ones which involve analysis at a molecular level with the use of DNA markers (Oliveira et al. 2005). Among the classes of available DNA markers, RAPDs (Random Amplified Polymorphic DNA) have been used in several studies to confirm interspecific hybrids, such as the ones performed among species of the genus Theobroma (Faleiro et al. 2003), Carica (Magdalita et al. 1997) and recently in Passiflora species (Junqueira et al. 2008). Simple Sequence Repeats (SSR) have been used in several studies involving paternity analysis, as in Prunus species (Schueler et al. 2003). This study aims to confirm the occurrence of crossed fecundation in F1 interspecific hybrids obtained through the crossing of P. sublanceolata J. M. MacDougal (ex P. palmeri var. sublanceolata Killip) versus P. foetida var. foetida L. using RAPD and SSR markers, describing the new hybrids based on morphological characters and analyzing their chromosome numbers and chromosomal homology.

Materials and methods Germplasm The experiment was performed with species P. sublanceolata (Killip) J. M. MacDougal (2n = 22; Abreu 2008) donated by Empresa Brasileira de

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Pesquisa Agropecua´ria (EMBRAPA) and P. foetida var. foetida L. (2n = 22; Bowden 1945) donated by Universidade Estadual do Norte Fluminense (UENF), Darcy Ribeiro campus at Campos dos Goytacazes, RJ. The plants were kept in the Passiflora Active Germplasm Bank from Universidade Estadual de Santa Cruz (UESC), Ilheús, Bahia, Brazil (long 39° 100 W, lat 14° 390 —S, altitude 78 m). Interspecific hybridizations The interspecific crosses were performed in a greenhouse in January 2006 with temperature and relative humidity ranging of 25–30°C and 70–90%, respectively, always from 7.30 to 9.00 a.m., a period in which the flowers remain open and the stigmas are receptive. Five plants of each species were used, P. sublanceolata as female parents and P. foetida var. foetida as male parents. The flower buds of genitors in pre-anthesis stage were protected with white paper bags one day before the hybridization, and the ones belonging to the female parent were emasculated. The paper bags were carefully placed over the buttons and sealed with adhesive tape at the floral peduncle, thus avoiding contamination with undesired pollen carried by insects. On the following morning, the anthers of P. foetida var. foetida were collected in bulk and carefully scrubbed over the stigmas of P. sublanceolata. After the artificial pollination, the flowers were labeled and again protected with paper bags for 24 h to avoid pollen contamination. The fruits resulting from such hybridizations were protected with nylon nets until they were ripe (Bruckner and Otoni 1999). The time between pollination and fruit fall was approximately 70 days. Five fruits were obtained, with 8–32 seeds per fruit. Approximately 80 seeds were planted in the substrate consisted of washed sand and bovine manure (1:1). Of this total, twenty putative hybrids survived and were kept in a greenhouse (6.0 9 7.0 9 39.5 m) with a semi-arched structure, covered with plastic additive against ultra-violet rays and 30% shade. During the period they remained in the greenhouse, temperatures and relative humidity ranged from 22 to 36°C and 60 to 93%, respectively. The plants were kept under these conditions for about 40 days before field trials. The hybrids were designated HD13 and each hybrid received specific numbers: HD13-101, HD13-102, HD13-103, HD13-105, HD13-107, HD13-115, HD13-123, HD13-124, HD13-125,

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HD13-133, HD13-134, HD13-135, HD13-136, HD13137, HD13-139, HD13-140, HD13-141, HD13-145, HD13-146 and HD13-147. DNA extraction Genomic DNA was extracted from young leaves, obtained from parent plants P. sublanceolata and P. foetida var. foetida aged 12 months, and from putative hybrids using the CTAB method (Doyle and Doyle 1990) with some modifications (Viana et al. 2006). Equivalent quantities of 250 ll DNA constituted a representative bulk of male parents, P. foetida var. foetida, and only one sample from a female parent, P. sublanceolata; one sample from each hybrid (HD13-101, HD13-133 and HD13-136, the most representative genotypes from each group of hybrids) were used. The samples of each genomic DNA were separated using electrophoresis in 0.8% agarose gel to estimate the concentration, integrity and purity of the extracted DNA. The gels were stained with ethidium bromide. After quantification, good quality DNA samples were diluted at a concentration of 10 ng ll-1. Obtaining RAPD markers The amplification reactions for seven RAPD primers (Table 1) were done in a total volume of 25 ll. The reaction mix contained 10 mM (pH 8.3) Tris–HCl, 50 mM KCl, 2.5 mM MgCl2, 150 lM of each one of the DNA, 0.2 mM of each decamer primer, which were from an Operon kit (Operon Technologies Inc., EUA), two units of enzyme Taq DNA Polymerase (Invitrogen, USA) and approximately 30 ng of DNA. The amplifications followed the program: one cycle at 94°C for 5 min; 35 cycles at 94°C for 1 min, at 32°C Table 1 Sequence of the seven RAPD decamer primers used to confirm crossed fecundation with the respective number of bands obtained for female and male parents, both parents, and the total number of bands (TNB) a

Primers from University of British Columbia (UBC)

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for 1 min, and at 72°C for 2 min; one cycle at 72°C for 7 min; and reduction at 4°C for 1 h using a thermocycler GeneAmp PCR System 9700 (Applied Biosystems, USA). After the amplification, the samples were run on 1.2% agarose gel. The electrophoretic separation took approximately 3 h at 110 V. The gels were stained with ethidium bromide, and the results were photo documented using an EDAS 290 (Kodak, USA) image analysis system under a UV transilluminator. Molecular weight markers (123 bp Ladder; Invitrogen, USA) were used to estimate the weight of the bands. Obtaining SSR markers and analysis Amplification reactions for microsatellite markers were performed in a total volume of 25 ll, containing 10 mM (pH 8.3) Tris–HCl, 50 mM KCl, 2 mM MgCl2, 200 lM of each one of the desoxinucleotides dATP, dTTP, dGTP and dCTP (100 mM, Bionovagency Biotecnologia e Comercio Ltda, Brazil), 0.3 lM of a decamer primer, one unit of Taq polymerase enzyme (5 u ll-1, Biotools B&M Labs. S.A., Spain) and approximately 30 ng of DNA. Previously, we tested cross-amplification of primers originally developed for Passiflora alata Curtis (Pa´dua et al. 2005) to the species involved in the hybridization. To confirm the cross-fertilization, cross-amplified single primer for both species (A08FP1- F: 50 -cacatttgccgtcactgg-30 , R: 50 -cggcatacgataaatctcctg-30 ) was used. After an initial denaturation at 95°C for 5 min, PCR conditions were: a 12-touchdown step proceeding at 95°C for 40 s, annealing beginning at 60°C until 54°C (decreasing 0.5°C per cycle) for 40 s. Twenty cycles at 95°C for 40 s, 54°C for 40 s and 72°C for 40 s were then performed, and a final extension was carried out at 72°C for 4 min. The PCR products were resolved on

Primera

Sequence 50 –30

Female parent

Male parent

Both parents

TNB

UBC03

50 -CCTGGGTCCA-30

3

2

0

5

UBC04

50 -CCTGGGTGGA-30

3

4

3

4

UBC06 UBC11

50 -GCCCGGTTTA-30 50 -CCGGCCTTAC-30

4 6

5 3

4 2

5 7

UBC17

50 -CTACCCGTGC-30

4

4

3

5

UBC23

5 -GTCCACACGG-3

0

4

3

2

5

UBC25

50 -ACCCCCGCCG-30

4

4

4

4

0

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6% denaturing polyacrylamide gels, and bands visualized by silver nitrate staining. The images were obtained using a Powerlook 1120 UDS Scanner (Amersham Biosciences, USA). Allele fragment lengths were calculated using a DNA size standard, 50 bp Ladder (1,000 lg ml-1, New England Biolabs, USA). Molecular markers generated by the primer were analyzed regarding the presence or absence of the parent bands in the progeny for confirmation of crossed fecundation. Field cultivation and morphological description of the hybrids After germination, the plants were transferred to black plastic bags containing soil and manure (2:1), until they reached around 1.0 m. After three months they were transferred to vases with a 42 l capacity, filled with sandy-argillaceous A-horizon soil, and then transferred to field where they were kept in of temperature and relative humidity conditions ranging from 18 to 39°C and 60 to 99%, respectively, during 18 months. They were pruned weekly and fertilized every 90 days with 3.9 g urea, 34.29 g mono-ammonium phosphate (MAP) and 14.97 g potassium chloride, as well as a solution containing micronutrients (1.01 g l-1 Bo; 2.5 g l-1 Cu; 0.16 g l-1 Mo; 8.43 g l-1 Zn; 5.58 g l-1 Mn) and urea (23.3 g l-1) every 15 days (Manica 1981). Due to the inexistence of morphological descriptors for ornamental passion fruits, we chose to adopt characteristics based on the floral and vegetative morphology, and the following aspects were observed for the description: (1) flower diameter; (2) corona filaments diameter; (3) flower peduncle length; (4) length of the first and second external series of corona filaments; (5) length and width of the petal; (6) length and width of the sepal; (7) internode length; (8) stem diameter of the primary, secondary and tertiary branches; and (9) length and width of the leaf (mm). Measurements were taken with the aid of a digital pachymeter (Stainless Steel Vernier Caliper—China). The variables were analyzed through descriptive statistics, with estimates for the general average and the total amplitude (maximum and minimum values). We also observed the qualitative characteristics of flowers such as the colors of stamen, style, stigma and anther, petal, sepal, corona filaments and leaves, using a Munsell Color Chart for Plant Tissue (Munsell Color Company 1977). The petal, sepal and leaf shape, as

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well as the consistency of the leaf and the presence or absence of trichomes were also observed. Cytogenetic analyses Cytogenetic analyses were performed after morphological description of the hybrids. Then, samples were obtained from three groups of hybrids (P. ‘Alva’, P. ‘Aninha’ and P. ‘Priscilla’) and from parents. To count chromosomes, young tips of roots were pretreated with 0.002 M 8-HQ at 4°C for 22 h, fixed in freshly made Carnoy fluid (ethanol-acetic acid, 3:1; Johansen 1940) for 2 h at room temperature and kept in fixative at -208 for at least 24 h. After they were washed, the samples were hydrolyzed in 5 N chloridric acid for 20 min at room temperature and transferred to Schiff reagent for 1 h, in the dark. The slides were prepared using the squash technique and the cells were stained with 1% acetic carmine. For the meiotic studies, flower buds in different development stages were fixed in Carnoy fluid for 3 h at room temperature, after three fixative changes within such period, and they were then stored at -20°C. Temporary slides were prepared using the squash technique and the cells were stained with 1% acetic carmine. To count chromosomes, ten metaphases were observed from each group of hybrids and parents. To observe the chromosomal homology, at least 50 meiocytes were analyzed in diakinesis (prophase I) and anaphase.

Results Interspecific hybridizations Interspecific hybridizations between P. sublanceolata and P. foetida var. foetida were successful. All the fruits obtained from crossings produced fertile seeds. The flowers of the parents and some hybrids are presented in Fig. 1. RAPD and SSR markers Out of the seven RAPD primers used, the primer UBC11 (50 - CCGGCCTTAC-30 ) generated informative bands used to confirm the crossed fecundation. Table 1 shows the number of bands present in female and male parents, number of bands shared by both parents and total number of bands (TNB), which is the

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Fig. 1 Parents and hybrids obtained out of the crossing P. sublanceolata 9 P. foetida var. foetida: a P. sublanceolata; b P. foetida var. foetida; c, d P. ‘Alva’; e, f P. ‘Priscilla’; g–l P. ‘Aninha’

sum of the number of bands present in female and male parents subtracted from the number of bands shared by both parents. Figure 2 shows the DNA amplification products: the male informative band of 123 bp was present in all the samples of the F1 progeny, confirming the occurrence of hybridization. The SSR primer A08FP1 amplifies species-specific fragments regarding the species involved in the cross (P. sublanceolata a 240 bp band and P. foetida var. foetida a 280 bp band). Molecular markers generated by the primer were analyzed in the progeny regarding the presence or not of the expected bands for confirmation of the crossed fecundation. Hybrids showed a heterozygote status with the two parent bands of 240 and 280 bp (Fig. 3).

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Morphological description of the hybrids The F1 hybrids demonstrated great variability of colors, shapes and sizes. They have herbaceous habits, represented by plants with stems that are not woody and can be grown in pots. The morphological characterization of the hybrids allowed differentiating into three groups registered by the Passiflora International Society (2008) as P. ‘Alva’ (#120), P. ‘Aninha’ (#121) and P. ‘Priscilla’ (#122). Hybrids HD13 136, HD13 123, HD13 115, HD13 135 and HD13 141 showed characteristics from the Passiflora ‘Alva’ group (coded as HD13 136), composed of plants with white flowers, large corona, light purple filaments at base, white and purple/white banding to apex. Hybrids

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Fig. 2 RAPD profile of P. sublanceolata, P. foetida var. foetida and putative F1 hybrids using primer 11 (50 -CCGGCCTTAC30 ). M: molecular weight marker 100 bp DNA ladder; f: P. sublanceolata (female parent); m: P. foetida var. foetida (male parent); 1: P. ‘Aninha’; 2: P. ‘Alva’; 3: P. ‘Priscilla’. The arrow indicates the male informative band to confirm the interspecific hybrids

Fig. 3 SSR profile using primer A08FP1. M: molecular weight marker 50 bp DNA ladder. f: P. sublanceolata (female parent); m: P. foetida var. foetida (male parent); Hybrids: 1. P. ‘Aninha’, 2. P. ‘Alva’, and 3. P. ‘Priscilla’

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HD13 105, HD13 101, HD13 102, HD13 107, HD13 147, HD13 124, HD13 137, HD13 125 and HD13 103 showed characteristics from the P. ‘Priscilla’ group (coded as HD13 101) with white flowers, small corona, dark purple filaments at base, white and purple to apex. Hybrids HD13 133, HD13 134, HD13 139, HD13 140, HD13 146, HD13145 showed characteristics from the P. ‘Aninha’ group (coded as HD13 133), pale pink flowers, corona filaments with a reddish/purple base, white, purple/white banding to apex. Passiflora ‘Alva’ (Fig. 1c, d). Individuals belonging to the P. ‘Alva’ group display the following characteristics: pilose vine, cylindrical stem 0.2–0.6 cm in diameter, 2.6–8.0 cm long internodes; threelobed, membranaceous leaves, hairy on both sides, 9.1–11.8 cm long, 7.1–9.0 cm wide, in shades of dark or pale green (4/4 7.5GY to 3/4 7.5GY); stout peduncle 4.6–9.2 cm long; showy white flowers, 5.8–7.6 cm in diameter, reflexing at anthesis (before midday); petals 2.5–3.4 cm long, 0.8–0.9 cm wide, white on both surfaces, apex ovate-lanceolate; sepals 2.7–3.3 cm long and 0.8–0.9 cm wide, white adaxial surface, green abaxial surface (4/8 7.5GY), ovate-lanceolate apex; corona filaments in 4 series, 3.7–4,6 cm in diameter, outermost series 1.3–1.6 cm long and second series 1.5–1.9 cm long, filiform, purple (4/4 5RP) at base, white and purple/white banding to apex; stamen filaments pale green; anthers adaxial surface pale green with yellow edges and pollen pale yellow; style slender, pale green; stigma olive green; fruits pendulous, ripening to scarlet; flowering all year in Brazil. P. ‘Priscilla’ (Fig. 1e, f). Individuals belonging to the P. ‘Priscilla’ group present the following characteristics: vine pilose, stem cylindrical 0.3–0.7 cm in diameter, internodes 4.4–9.5 cm long; leaves threelobed, membranaceous, hairy on both sides, 9.5–12.1 cm long, 7.5–9.0 cm wide, in shades of dark or pale green (4/4 7.5GY to 3/4 7.5GY); peduncles stout, 5.0–7.2 cm long; showy and white flowers, 5.6–7.1 cm in diameter, reflexing at anthesis (before midday); petals 2.5–3.0 cm long and 0.9–1.1 cm wide, white on both surfaces, apex ovate-lanceolate; sepals 2.2–3.1 cm long, 0.9–1.1 cm wide, adaxial surface white, abaxial surface green (4/8 7.5GY), apex ovate-lanceolate; corona filaments in 4 series, 2.8–3.9 cm in diameter, outermost series 0.9–1.4 cm long, second series 1.0–1.5 cm long, filiform, purple at base, white and purple (3/6 5RP) to apex; stamen filaments pale

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green; anthers adaxial surface pale green with yellow edges and pollen pale yellow; styles slender, pale green; stigma olive green; fruits pendulous, ripening to scarlet; flowering all year in Brazil. P. ‘Aninha’ (Fig. 1g–l). Individuals belonging to the P. ‘Aninha’ group present the following characteristics: vine pilose, stem cylindrical 0.4–1.1 cm in diameter, internodes 6.2–11.0 cm long; leaves threelobed, membranaceous, hairy on both sides, 8.7–11.6 cm long, 6.6–8.3 cm wide, in shades of dark or pale green (4/4 7.5GY to 3/4 7.5GY); peduncles stout, 4.3–6.7 cm long; flowers pale pink (8/4 5RP to 7/8 5RP), 6.0–7.0 cm in diameter, reflexing at anthesis (before midday); petals 2.5–2.9 cm long, 0.9–1.2 cm wide, pale pink on both surfaces (8/4 5RP to 7/6 5RP), apex ovate-lanceolate; sepals measuring 2.6–3.1 cm long, 0.9–1.2 cm wide, adaxial surface pale pink and sometimes darker than the petals (8/6 5RP to 7/8 5RP), abaxial surface green (4/8 7.5GY), apex ovate-lanceolate; corona filaments in 2–4 series, 2.9–3.6 cm in diameter; outermost series 1.0–1.4 cm long, second series 1.1–1.5 cm long, filiform, reddish/purple (3/6 5RP to 3/4 5RP) base, white, purple/white (3/8 5RP to 3/6 5RP) banding to apex; stamen filaments pale green; anthers adaxial surface pale green with yellow edges and light yellow pollen; style slender, pale green; stigma olive green; fruits pendulous, ripening to scarlet; flowering all year in Brazil. Cytogenetic analyses The chromosome number 2n = 22 was analyzed in the parents (Fig. 4). In the hybrids P. ‘Alva’ and P. ‘Priscilla’, both with white petals and sepals, 2n = 20 was observed, while the hybrid P. ‘Aninha’, with pale pink petals and sepals, presented 2n = 22. Meiosis was regular in parents and hybrids. More than 90% of the cells were bivalents at diplotene and diakinesis, and more than 87% of the meiocytes analyzed in anaphase I and II presented normal segregation (Fig. 4).

Discussion The progeny population obtained by crossing P. sublanceolata and P. foetida var. foetida segregated for several characteristics, particularly for floral characteristics. In the hybridization P. sublanceolata

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Fig. 4 Cytological analysis in parents and hybrids obtained out of the crossing P. sublanceolata 9 P. foetida var. foetida. a P. foetida (2n = 22); b P. sublanceolata (2n = 22); c P. ‘Aninha’ (2n = 22); d–f P. ‘Priscilla’—d 2n = 20; e Diakinesis (n = 10); f Anaphase I without irregularities. Bar = 5 lm

versus P. foetida var. foetida, twenty genotypes were obtained, and the variation could clearly be classified in three groups, determining three varieties: P. ‘Alva’, P. ‘Priscilla’ and P. ‘Aninha’. Sexual hybridization with Passiflora can be successfully performed, both between plants of the same species and among related species, only some combinations are not successful (Torres and Martin 1974). Generally closest species, such as P. sublanceolata and P. foetida, do not present these barriers. In the studied passion flowers, the success obtained in the hybridization is probably due to the fact that such species belong to the same taxonomic classification (Dysosmia), and the same has happened in other genera such as Capsicum (Naci Onus and Pickersgill 2004) and Cajanus (Thiruvengadam and Muthiah 2007). Most of the time, the closest species have the same chromosomal number, and thus are crossed more easily, as was observed with P. sublanceolata 9 P. foetida var. foetida and P. edulis 9 P. setacea (Soares-Scott et al. 2003). The combining ability, a very important factor for improvement programs, is also a consequence of the association between favorable alleles and a regular meiotic process, so that it gives origin to the formation of viable gametes (Defani-Scoarize et al. 1996). Passiflora ‘Aurora’ obtained by Maurizio Vecchia (Ulmer and MacDougal 2004) is a known hybrid from P. foetida versus P. sublanceolata, but there is no information as to what variety of Passiflora foetida was used. Passiflora ‘Aurora’ is different from such

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plants because they present slightly bigger flowers (7.5–8 cm) pink to purplish, white and violet; sepals (3.4–4 cm) greenish pink outside, pink to purplish inside and petals pink to purplish, sub-equal to sepals, least corona filament series (from 2 to 3), however, the colors are similar to hybrids in this study, white with purplish violet base and violet apex (Ulmer and MacDougal 2004). Using the same parents, the hybrid P. ‘Pink Jewel’ obtained by R. J. R. Vanderplank is different from such plants because they present more corona filament series (from 5 to 6), slightly bigger sepals and petals (3.6–3.8, 3.7–4.2 cm, respectively), but in terms of flower colors, the description is not detailed (Vanderplank 2000). RAPD and SSR markers based on the analysis of informative bands was a successful methodology to confirm hybridization in HD13 progeny. According to Faleiro et al. (2003) the use of one or two primers or of primer combinations with at least one informative band is enough to confirm the occurrence, or not, of crossed fecundation. Each informative band works as a marker gene habitually used by plant breeders (Bore´m 1997). RAPD markers were used to confirm the crossed fecundation between two fructiferous plants of great economical importance, Theobroma cacao and T. grandiflorum (Faleiro et al. 2003). Reproductive studies in Prunus species involving SSR markers were based on estimates of probability of paternity exclusion (Schueler et al. 2003). Several crossings among Passiflora species were confirmed using RAPD markers, such as P. setacea versus P. coccinea (Junqueira et al. 2005, 2008). Junqueira et al. (2008) obtained 17 interspecific hybrids and RAPD markers were utilized to confirm crosses such as P. glandulosa versus P. sidaefolia and P. caerulea and P. amethystina, and according to the authors, the species presented genetic compatibility. Crossing confirmations among Passiflora species using SSR markers in Passiflora were not found. The direction and the facility with which two species may be crossed, and the chromosome behavior during the meiosis of parents and hybrids are considered important criteria to evaluate the degree of genetic proximity among species and accessions (Rao and Rao 1984). The meiotic behavior of the hybrids was regular. This fact indicates genetic compatibility between parents and probably fertile hybrids, since the meiotic behavior of a plant is closely related to its

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fertility degree (Defani-Scoarize et al. 1996). The deletion (2n = 22 - 2) observed in hybrids P. ‘Alva’ and P. ‘Priscilla’ did not negatively affect the development of plants, so such chromosomes are probably made of repetitive sequences and of non-deleterious genes. The new genome constitution produced by the hybridization process may generate intergenome conflicts, leading to genetic rearrangements (Riddle and Birchler 2003), like individual chromosomal eliminations observed in the hybrids of F1 progeny HD13. Some hypothesis to the chromosomal elimination process may be thought, like the inactivation of chromosomes by nucleases, degradation of chromatin, suppression of the centromere function and asynchrony of the cell cycle phases (Singh 2002), being also subject to the influence of environmental factors (Linde-Laursen and Von Bothmer 1999). Aneuploidy occurs often due to errors as a result of meiotic nondisjunction during the first or second meiotic divisions (Singh 2002). Genes involved in transmitting signals to the formation of the spindle fibers are equally important for the harmonious course of meiosis (Caetano-Pereira and Pagliarini 2001), correctly guiding the chromosomes in the metaphase plate and leading to perfect segregation to the poles. Genetic alterations or environmental factors may influence the expression of genes during meiosis (Souza et al. 2003), and cause failures in fiber formation and affect chromosome disjunction (Palmer et al. 1992; Caetano-Pereira and Pagliarini 2001). The combined action of dv genes in abnormal meiotic spindle orientation in Zea mays L. (Caetano-Pereira and Pagliarini 2001; Shamina et al. 2000) and interspecific hybrids of Brachiaria (Mendes-Bonato et al. 2006), besides synaptic mutants and disjunction and chromosome segregation, support the hypothesis of alteration in the expression of these genes as a possible cause of these irregularities. Considering the above statements, the parents probably share alterations in these genes. In this case, the processes of irregular segregation and nondisjunction common to parents P. sublanceolata and P. foetida would be related to the aneuploidy process observed in F1 hybrid progeny. Meiotic analysis preceding this study indicated several chromosome numbers for P. foetida, n = 10 (Beal 1969) and n = 11 (Bowden 1945; Storey 1950), corroborating this study.

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Conclusions The interspecific hybridization P. palmeri var. sublanceolata 9 P. foetida var. foetida was successful mainly due to the genetic compatibility of the two parents, confirmed by the chromosomal homology observed in the cytogenetic analysis. Of the 20 hybrids characterized, three different varieties of ornamental interest were selected, P. ‘Alva’, P. ‘Priscilla’ and P. ‘Aninha’, and their hybridity was confirmed at DNA level, using RAPD and SSR markers. One marker was enough to verify the occurrence of crossing between the two species P. sublanceolata and P. foetida var. foetida, thus demonstrating to be a trustworthy, fast and efficient methodology. Some hybrid plants of progeny HD13 supported the loss of one chromosome pair. The hybrid plants that presented the aneuploidy showed white flowers, but other studies are necessary to understand this relation. Acknowledgments The authors wish to thank Programa de Po´s-Graduaçaõ em Produçaõ Vegetal and Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES) for the scholarship of the first author; Fundaçaõ de Amparo a` Pesquisa do Estado da Bahia (FAPESB), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Universidade Estadual de Santa Cruz (UESC) for financially supporting the research; Dr. Armando Carlos Cervi (Universidade Federal do Parana´—PR, Brazil) and Dr. Luiz Carlos Bernacci (Instituto Agronoˆmico—Campinas, SP, Brazil) for the taxonomic identification of parents.

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